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The case against embryonic stem cell research: an interview with yuval levin.

Scientists largely agree that stem cells may hold a key to the treatment, and even cure, of many serious medical conditions. But while the use of adult stem cells is widely accepted, many religious groups and others oppose stem cell research involving the use and destruction of human embryos. At the same time, many scientists say that embryonic stem cell research is necessary to unlock the promise of stem cell therapies since embryonic stem cells can develop into any cell type in the human body.   

In late 2007, researchers in the United States and Japan succeeded in reprogramming adult skin cells to act like embryonic stem cells. The new development offers the possibility that the controversy over the use of embryos could end. But many scientists and supporters of embryonic stem cell research caution that this advance has not eliminated the need for embryos, at least for the time being.

Recently, the Pew Forum sat down with Yuval Levin, author of Tyranny of Reason , to discuss the ethical and moral grounds for opposing embryonic stem cell research. Previously, Levin was the executive director of the President’s Council on Bioethics. Currently, he is the Hertog Fellow at the Ethics and Public Policy Center in Washington, D.C., where he also directs the center’s Bioethics and American Democracy program.

A counterargument   explaining the case for embryonic stem cell research is made by Jonathan Moreno, a professor at the University of Pennsylvania and a senior fellow at the Center for American Progress in Washington, D.C.

Featuring : Yuval Levin , Hertog Fellow and Director of the Bioethics and American Democracy Program, Ethics and Public Policy Center

Interviewer: David Masci , Senior Research Fellow, Pew Forum on Religion & Public Life

Question & Answer

Recently, researchers in the United States and Japan successfully turned human skin cells into cells that behave like embryonic stem cells. There has been some discussion that this advance makes the moral and ethical debate over embryonic stem cells moot. Do you think that’s an accurate assessment?

I think it’s going to take a while for the ethical debate to catch up with the science. The scientific community has reacted very positively to this advancement, which was made in November 2007. There have been many additional scientific studies published on the topic since then, and it appears increasingly likely that the cells produced using skin cells are the equivalent of embryonic stem cells. So I think that, in time, this probably will be the final chapter of this particular debate about embryonic stem cells, but I don’t think we’re at the end of it quite yet.

Do you agree with Professor James Thomson, who led the American research team that made this breakthrough, when he maintains that this advance does not, for the time being, abrogate the need for embryonic stem cell research?

Part of his argument for continuing to use embryonic stem cells was backward-looking to make the point that researchers wouldn’t have been able to develop this technique if they hadn’t been doing embryonic stem cell research. I think that’s true, although in a certain way it actually vindicates the logic of President Bush’s stem cell policy, which is to allow some work to be done – without creating an incentive for the destruction of further embryos – to advance the basic science in these kinds of directions.

Thomson also argued that there will still be a need to use embryos in the future. I think that’s also a fair argument in the sense that there are always interesting things to learn from different kinds of experiments, but it doesn’t address the ethical issues surrounding the debate. If there were no ethical concerns, then certainly the new development wouldn’t mean embryonic research would become totally useless. But given that there are concerns, the case for destroying embryos does become a lot weaker. For some people, myself included, the ethical concerns are matters of principle and don’t change with new developments.

But for a lot of people, the stem cell debate has always been a matter of balance. People are aware that there are ethical concerns and that there is enormous scientific promise. Now the debate is: Given the ethical questions at stake, is the scientific promise sufficient to make us put the ethical concerns aside and support the research? I think that balance has changed because of this advance, and having an alternative to embryonic stem cell research that achieves the same result will obviously affect the way people think about the ethics of this issue.

That doesn’t mean the scientists no longer have any use for embryonic stem cells or even that they won’t have any use for them. But I do think it means that people are going to change the way they reason about the balance between science and ethics because of this advance.

I know that you believe that human embryos have intrinsic worth. Do you believe that they have the same intrinsic worth as a five-year-old child or a 50-year-old man?

The question of intrinsic worth is complicated. I don’t think it is right to try to determine an embryo’s intrinsic worth by debating when human life begins. The question of when life begins is a biological question, and the answer actually is fairly straightforward: The life of an organism begins at conception. The ethical question, however, is not about when a life begins but whether every life is equal, and that’s a very different question.

I think that the embryonic stem cell debate is ultimately about the question of human equality. The United States has had one answer to that question written in its “birth certificate” – the Declaration of Independence – which states that “all men are created equal.” I think that examining this principle of human equality provides the right answer to this debate, but it is not a simple answer. Human equality doesn’t mean that every person is the same or that every person can even be valued in the same way on every scale. What it means is that our common humanity is something that we all share. And what that means, in turn, is that we can’t treat a human being in certain ways that we might non-human beings.

The protection of human life comes first. And to the extent that the debate is about whether it is acceptable to destroy a living human being for the purpose of science – even for the purpose of helping other human beings – I think that in that sense, the embryo is our equal. That doesn’t mean that I would think of an embryo in the same way that I would think of a three-year-old child, but I would reject a technique that uses either of them for scientific experimentation.

So in other words, even though you would grieve the death of a 50-year-old man more than a five-day-old embryo, on at least the most basic level you believe that they both have the same right to life.

Yes, that’s right. And right to life derives from human equality. The right to life is, in a way, drawn out of the political vocabulary of the Declaration of Independence. And so, to my mind, the argument at the heart of the embryonic stem cell debate is the argument about human equality.

Recently in The New Republic magazine, Harvard psychologist Steven Pinker wrote that conservative bioethicists like yourself consistently predict the worst when looking at developments in biotechnology. He went on to say that had there been a president’s council on cyber-ethics in the 1960s, “no doubt it would have decried the threat of the Internet since it would inexorably lead to 1984 or computers ‘taking over’ like HAL in 2001 .” How do you respond to this suggestion that there always seems to be this sort of chorus of doomsayers every time something new comes along?

To my mind, biotechnology is fundamentally different from past developments in technology because it’s directed to the human person. From the beginning of the scientific revolution, science and technology have tried to allow us to manipulate and shape the world around us for the benefit of man. Now that we’re beginning to manipulate and shape man, the question is: For the benefit of what? In some cases that’s easy to see. Obviously curing disease is more of an “old-fashioned” scientific pursuit. But there are newer scientific developments, such as certain types of human enhancement technologies that raise very complicated questions of how we should judge the ends and the means of technological advancements. That being said, Pinker has a point, in a larger sense – that judging the risks of new technologies is very difficult. In general, I think we ought to give the benefit of the doubt to our ability to use new technologies. I don’t think that we should assume that the worst will happen. But there are specific instances, which are few but very important, when we do need to be cautious.

Let’s shift gears to a question about religion and faith. Obviously there are people of faith on both sides of this debate. In fact, there are conservatives – traditional social conservatives, such as Republican Sen. Orrin Hatch of Utah – who support embryonic stem cell research. But could you explain how the Judeo-Christian and Western moral ethic informs your views on this issue and why you think that God is ultimately on your side?

Well, I don’t know that I think that. My approach to this is not religious. I’m not a particularly religious person and I come at this from more of a liberal democratic concern for human equality and the foundations of our society. That being said, those foundations are not utterly secular, and my understanding of them is not utterly secular. I think that to believe in human equality you do have to have some sense of a transcendent standard by which to make that judgment. In other words, when we talk about equality, what do we mean? Equal in relation to what?

Some people have certainly tried to make a purely secular liberal argument for human equality. While I think it’s very hard to ground a genuine, deep belief in human equality in a worldview that sees nothing above the material, I don’t think that that belief depends on specific theological commitments. To my mind, it’s an American belief more than it is a religious belief.

Certainly I think that President Bush’s commitment to human equality has a lot to do with a particular Christian sense of human worth and human value. But I don’t think that it’s necessary to ground yourself in a particular theological or sectarian preference. I think that this is really about whether we believe in a liberal society, which comes from a belief in human equality. The American left, which for the most part is on the other side of this debate from where I am, has always been the champion of human equality, and I think that it’s a question that they have to really think about.

The Pew Forum and the Pew Research Center for the People & the Press have done polling on this issue over the last six or seven years and have found that Americans generally favor embryonic stem cell research. Why do you think this has happened, and what do you think this trend indicates?

That’s an interesting question. We actually did a poll here at the Ethics and Public Policy Center in February on a similar question, and the lesson I drew from that, and from some other polling that’s been done, is that on the stem cell debate, people are just very confused about the facts, and the trend lines have generally followed the sense that cures are coming. In the end, the issue has been misrepresented as a choice between cures and Christianity, and people increasingly think that curing people like Christopher Reeve is just as much of a human good as protecting an embryo that they can’t even imagine.

But when you dig down into people’s views about stem cell research, you find a great deal of confusion, and when you put the questions in ethical terms, you find small majorities opposing it. When you put the question in medical terms, you find, I think, somewhat larger majorities supporting it. In our poll, we asked the same people a series of questions that basically put the same issue in several different ways, and their responses are total opposites of one another. The fact that the same people come out on the opposite sides of the same issue when it’s put in different ways suggests to me that the issue is very hard to understand – which it is.

Frequently one hears that, ultimately, you can’t stop science or “progress” and that ethical, moral and religious objections inevitably will fall by the wayside when there are clear material gains to be made. Do you think that’s the most likely scenario in this case, assuming the scientific community continues to see a need for embryonic stem cell research?

Well, that’s the big assumption, right? To my mind, the aim of people such as myself has always been to find ways of doing the science without violating the ethics rather than to force a choice between the science and the ethics. If we force that choice, I think it’s more likely that the country would choose science over ethics, and that’s exactly why we have to avoid the choice. I don’t think we should be overconfident in our ability to persuade people to pass up a material benefit for an ethical principle, although I hope that can be done in the stem cell research debate. It certainly has been done in some instances when the principle was more evident and more obvious – such as imposing limits on human subject research.

Again, the aim from my point of view – and from a lot of people on my side of this argument – has been to find ways to advance the science without violating the ethics. That’s the logic of President Bush’s stem cell policy; that’s why people have been pushing for alternatives; that’s why they’re encouraging the development of these latest alternatives – to avoid the choice, not to force the choice. I think that’s the best thing for the country, from everybody’s point of view. You don’t want a situation where you’ve got sort of red-state medicine and blue-state medicine and people believe that the treatment their hospital is giving them is obtained in unethical ways. That would begin to break up the practice of medicine and to affect our attitudes about science – which on the whole has done a tremendous amount of good for society. So I think what everybody should aim for is finding a way to end this potentially very damaging debate rather than force a choice.

This transcript has been edited for clarity, spelling and grammar.

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About Pew Research Center Pew Research Center is a nonpartisan fact tank that informs the public about the issues, attitudes and trends shaping the world. It conducts public opinion polling, demographic research, media content analysis and other empirical social science research. Pew Research Center does not take policy positions. It is a subsidiary of The Pew Charitable Trusts .

A regulatory argument against human embryonic stem cell research

Affiliation.

• 1 The National Catholic Bioethics Center, Philadelphia, PA 19151, USA. [email protected]
• PMID: 19690326
• DOI: 10.1093/jmp/jhp036

This article explores the plausibility of an argument against embryonic stem cell research based on what the regulations already say about research on pregnant women and fetuses. The center of the argument is the notion of vulnerability and whether such a concept is applicable to human embryos. It is argued that such an argument can be made plausible. The article concludes by responding to several important objections.

• Embryo Research / ethics*
• Embryo Research / legislation & jurisprudence*
• Embryonic Stem Cells*
• Philosophy, Medical*
• Vulnerable Populations / legislation & jurisprudence

• Open access
• Published: 24 February 2023

Mesenchymal stem cell-released oncolytic virus: an innovative strategy for cancer treatment

• Nadia Ghasemi Darestani 1 ,
• Anna I. Gilmanova 3 ,
• Moaed E. Al-Gazally 2 ,
• Angelina O. Zekiy 3 ,
• Mohammad Javed Ansari 4 ,
• Rahman S. Zabibah 5 ,
• Mohammed Abed Jawad 6 ,
• Saif A. J. Al-Shalah 7 ,
• Jasur Alimdjanovich Rizaev 8 ,
• Yasir S. Alnassar 9 ,
• Naseer Mihdi Mohammed 10 ,
• Yasser Fakri Mustafa 11 ,
• Mohammad Darvishi 12 &
• Reza Akhavan-Sigari 13 , 14  

Cell Communication and Signaling volume  21 , Article number:  43 ( 2023 ) Cite this article

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Oncolytic viruses (OVs) infect, multiply, and finally remove tumor cells selectively, causing no damage to normal cells in the process. Because of their specific features, such as, the ability to induce immunogenic cell death and to contain curative transgenes in their genomes, OVs have attracted attention as candidates to be utilized in cooperation with immunotherapies for cancer treatment. This treatment takes advantage of most tumor cells' inherent tendency to be infected by certain OVs and both innate and adaptive immune responses are elicited by OV infection and oncolysis. OVs can also modulate tumor microenvironment and boost anti-tumor immune responses. Mesenchymal stem cells (MSC) are gathering interest as promising anti-cancer treatments with the ability to address a wide range of cancers. MSCs exhibit tumor-trophic migration characteristics, allowing them to be used as delivery vehicles for successful, targeted treatment of isolated tumors and metastatic malignancies. Preclinical and clinical research were reviewed in this study to discuss using MSC-released OVs as a novel method for the treatment of cancer.

Video Abstract

Introduction

In recent years, early diagnosis of some cancer types, along with the development of cancer-specific treatments, has led to an increase in cancer patients' survival rates [ 1 ]. However, the short half-life of several cancer-specific medications, restricted distribution to particular tumor types, and negative impacts on healthy tissues are important barriers to treatment. Actually, the primary goal of cancer treatment is to develop anticancer medications that effectively target malignant cells while preserving healthy tissue [ 2 , 3 ]. A few instances of metastatic cancer have been effectively treated using traditional methods [ 4 ]. As a result, developing a novel therapeutic approach to inhibit metastasis is crucial, especially given the issues with current cancer treatment strategies, such as drug resistance and systemic side effects [ 5 , 6 ].

Beyond the capacity of some viruses to mediate oncogenesis and their use in the development of immunotherapies, such as the cytomegalovirus (CMV) gliomagenesis and its implications in the development of CMV-specific adoptive T cell immunotherapies, viruses themselves can be used as therapeutic agents to target tumor cells [ 7 ]. In this way, oncolytic viruses (OVs) are defined as naturally occurring or genetically manipulated viruses that exclusively replicate and grow in tumor cells and kill them while sparing normal cells [ 8 , 9 ]. Oncolytic viral therapy is a new strategy of cancer therapy that has shown promise in preclinical and clinical trials [ 10 , 11 ]. Altered mutants of human viruses, wild-type animal viruses that are cytotoxic to human cancer cells, and live virus vaccines are among the viruses used in this therapy. Adenovirus, measles virus, reovirus, herpes simplex virus, vesicular stomatitis, Newcastle disease virus, vaccinia virus, and poliovirus are some of these viruses [ 12 , 13 ]. OVs have the ability to directly lyse cancer cells but this is not the sole advantage of them; it is now well acknowledged that one of the most essential aspects of virotherapy is the cytotoxic immune response they can trigger or reactivate in patients, which results in therapeutic responses [ 14 , 15 ] and was shown in glioblastoma, B cell malignancy, metastatic melanoma, and liver cancer [ 16 , 17 , 18 , 19 , 20 ]. Indeed, multiple investigators have reported on the possible use of OVs for cancer treatment, with a demonstration of long-term prognosis [ 21 ]. A variety of factors, including viral elimination by the immune system and viral uptake by tissues and organs, can influence viral effectiveness in reaching cancerous tissues [ 22 , 23 , 24 ]. To boost treatment efficacy, effective carrier vehicles are essential for delivering OVs to tumor sites. Adult stem or progenitor cells have been extracted from a variety of tissues, including the brain, heart, and kidney, and have shown promise in treating a variety of diseases [ 25 , 26 , 27 ]. In both in vitro and many murine cancer models, unmodified MSC has been demonstrated to have anti-tumor activities. This is due to antitumor substances generated by MSCs, which limit the growth of cancer cells including glioma, melanoma, lung cancer, hepatoma, and breast cancer [ 28 , 29 , 30 , 31 , 32 , 33 ]. Furthermore, MSCs have been utilized as carriers because of their known tumor-specific homing ability, which allows for the virus's safe transportation and releases on the tumor site [ 34 , 35 , 36 , 37 ]. Using MSCs might be a method to enhance the quantity of oncolytic virus given to patients while reducing side effects and avoiding direct tumor injections [ 38 ]. Altogether, in this paper we will review the features of MSCs and OVs as well as their activities against tumors, and also discuss challenges in using this strategy as an innovative cancer treatment.

The features of the MSC-based delivery of OV

As a novel cancer treatment method, virotherapy offers various benefits, including the likely absence of cross-resistance with traditional treatments and the ability to promote tumor elimination through a variety of pathways. In addition, MSCs are suitable carriers for anticancer viruses since they can home to tumor sites [ 39 , 40 , 41 , 42 ], are easy to isolate and develop in vitro, and have strong metabolic activity, which is necessary for viral replication [ 34 , 43 ].

MSCs as OV carriers

The majority of preclinical research has discovered efficient features for MSCs as OV carriers [ 44 , 45 , 46 ] and this strategy might be an effective way to promote oncolytic virotherapy efficiency [ 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 ]. Indeed, MSCs have been shown to be one of the best alternatives for OV chaperoning in the treatment of cancer due to their tumor-homing, inherent anticancer capacities, OVs preservation from neutralizing antibodies, and viral distribution to the tumor site via the Trojan horse strategy [ 53 , 55 , 56 ].

In a series of studies, MSC has been discovered to migrate to the area of injury, ischemia, and tumor sites via chemotaxis [ 57 ]. Although the mechanisms by which MSC migrate across the endothelium or to targeting sites remain unclear, extensive research has demonstrated that MSC migration is regulated by the cytokine/receptor pairs, such as SDF-1/CXCR4, SCF/c-Kit, HGF/c-Met, VEGF/VEGFR, PDGF/PDGFR, MCP-1/CCR2, and HMGB1/RAGE [ 58 ]. In the TME, many immune cells, as well as cancer cells, release soluble molecules that can directly influence MSC chemotaxis to injured tissues. The oxidative state, vascularization, and inflammatory condition of the tumor can all influence MSC migration efficiency at these locations [ 59 ]. It has been shown that interleukin-6 (IL-6) can promotes MSC tropism to cancer sites [ 60 ] and also this movement is IL-8-dependent in glioma [ 61 ]. Tumors can also recruit MSCs from other tissues, such as bone marrow (BM-MSCs) and adipose tissue (AD-MSCs), and promote their engraftment into the TME through inflammatory signals [ 62 , 63 , 64 ]. Local variables including hypoxia, cytokines, and Toll-like receptor (TLR) ligands stimulate recruited MSCs to multiply and express growth factors that enhance tissue regeneration at the site of injury [ 65 ]. Hepatic carcinoma [ 66 ], breast cancer [ 67 ], and glioma have all been demonstrated to attract MSCs [ 68 ].

This therapeutic strategy, in addition to offering substantial site-specificity, avoids potential issues associated with biological drug half-life limitations, as drug release might be tailored to be constant [ 43 ]. Moreover, it's hard to manage effective concentrations of anti-tumor drugs near the tumor for long periods of time [ 69 ]. For instance, in the case of brain tumors, the failure of substances to cross the blood–brain barrier is a concern. The use of MSCs as cellular delivery vehicles has been proposed as a novel approach to addressing these obstacles, allowing for a more precise and long-lasting therapeutic response than standard delivery methods would ordinarily allow [ 2 ]. Another unique aspect of employing MSCs as OV carriers is that they may function as biological manufacturers for viral genome replication, enhancing virus titer. This implies that a low initial dose of OVs for loading into MSCs is sufficient to deliver a high viral dose to tumor microenvironment. However, the specifics of OVs replication within MSCs remain unknown [ 70 , 71 ]. As a result, when OVs are carried by MSCs, they leverage MSCs' natural affinity to reach tumor sites, improving OVs homing and promoting oncolysis.

Immunosuppressive activities of MSCs

Multiple studies revealed that MSCs have anti-inflammatory and immunosuppressive properties, so that, these cells generate and release a number of soluble cytokines, such as IL-6, IL-10, TGF-β1, heme oxygenase-1(HO-1), inducible nitric oxide synthase (iNOS), and indoleamine-2-dioxygenase-3(IDO) [ 72 ]. These cytokines are essential in immunosuppression and inhibiting B lymphocyte maturation and restricting their capacity to produce immunoglobulin [ 73 , 74 , 75 ], inhibiting the secretion of cytokines by helper T cells, reducing the cytotoxic actions of effector T lymphocytes [ 76 ], decreasing NK cell proliferation, cytotoxicity, and cytokine generation [ 77 ]. Furthermore, in a study on diabetic nephropathy in rats, it was shown that MSCs suppressed CD103+ DCs and CD68+ CD11c+ macrophages in the kidneys and alleviate renal injury [ 78 , 79 , 80 , 81 ]. They are also able to inhibit the differentiation of CD14+ monocytes and CD34+ progenitor cells into mature DCs [ 82 ], restrict DC differentiation and function [ 83 ], and thereby increase CD4 + CD25 + FOXP3+  T lymphocytes (Treg) development and generation of other regulatory immune subtypes, including CD8+ CD28−T lymphocytes [ 84 , 85 ], IL-10-producing B lymphocytes [ 86 ], and IL-10-producing DCs [ 87 ]. These actions are essential MSC characteristics for inhibiting local inflammation during virotherapy and permitting the oncolytic virus to replicate and destroy cancer cells without immune constraint. [ 88 ].

Anti-tumor effects of MSCs

Several investigations have indicated the specific homing of oncolytic virus-loaded MSCs to tumor xenografts and consequent infection of tumor cells, results in diminished tumor sizes and a considerable improvement in the survival rates of treated animals [ 89 , 90 , 91 , 92 , 93 , 94 , 95 ]. MSCs enhance the proliferation of some tumor cell lines in vivo but not others. Variations in tumor types, MSC preparations, duration, and quantity of MSC delivery may cause differences in the functional role of MSCs in tumor growth [ 96 ]. Indeed, MSCs are thought to limit tumor development by interrupting the cell cycle, reducing proliferation, inhibiting the PI3K/AKT pathway, and expressing suppressor genes [ 97 , 98 ]. In a breast cancer metastasis mouse model, the umbilical cord derived MSCs (UC-MSC) and AD-MSCs were administered, and it was shown that they could prevent lung metastasis and slow tumor development by cleavage of the poly (ADP-ribose) polymerase (PARP) and caspase-3, which could then trigger apoptosis [ 99 ]. Another study showed that murine bone marrow MSCs had a cytotoxic impact on the tumor in a melanoma murine model through the production of reactive oxygen species when in interact with endothelial cells located at the capillaries [ 100 ]. As a result, the tumor development was delayed and the endothelial cells undergo apoptosis. Nevertheless, the MSCs' cytotoxic effects were only apparent when they were implanted in large quantities [ 101 ]. In addition, in a mouse model of Kaposi sarcoma, human MSCs (hMSCs) administered intravenously (iv.) were found to home to carcinogenesis sites and potently decrease tumorigenesis. Cell contact via E-cadherin and Akt inhibition were essential for the suppression of the sarcoma cells proliferation [ 102 ]. MSCs have been also discovered to display anti-angiogenic properties in vitro and in melanoma murine models [ 101 ]. Moreover, AD-MSCs have both (pro- and anti-cancer) capabilities in breast [ 103 ] and prostate cancer [ 104 ].

Mechanisms of oncolytic virotherapy

Various mechanisms of action are used by oncolytic viruses. Selective replication inside tumor cells leads to a direct lytic impact on tumor cells and the development of a systemic anti-tumor immune response. Depending on the origin and kind of cancer cell, the viral vector's properties, and the interactions between the virus, TME, and host immune response, the proportional involvement of different mechanisms could differ [ 105 ]. Indeed, Oncolytic virotherapy depends on a balance of antiviral mechanisms that kill the virus and pro-immune mechanisms that detect cellular epitopes, TAAs, and neoantigens from virus-infected tumor cells [ 106 ]. In addition to their anti-tumor properties, OVs stimulate antiviral immunity against viral antigens from the resulting infection, which is a critical player during OV-based treatments due to its capability to create an advantageous microenvironment for the immune system's activity against specific cancer cell indicators [ 107 , 108 ].

The two major methods by which OVs destroy tumors are direct cell death and the activation of anti-tumor immunity [ 109 ]. The first strategy employs the virus's biological life cycle; oncolytic viruses may destroy cancer cells infected with them by direct virus-induced cytotoxicity, which is regulated by a variety of cytotoxic immune activation pathways. Following cell proliferation and lysis, virions infect surrounding cells and repeat the lytic cycle, enabling the treatment to self-amplifying at the site of need [ 110 ]. This cycle repeats until the virus's replication is reduced or the number of vulnerable host cells is decreased [ 111 ]. On the other hand, OVs are one of the most well-known immunogenic cell death inducers, and they're more probably to be type II stimulators than type I stimulators [ 112 ]. Although the precise mechanisms by which oncological viruses operate are elusive, it is suspected that they function by regulating the infected cancer cell's molecular cell death machinery. The majority of cancer cells may be resistant to apoptosis [ 113 ] and according to researchers, they have several apoptosis evasion mechanisms but they may be driven to die by non-apoptotic processes. Recent studies demonstrated that OVs can induce immunogenic cell death (ICD) in cancer cells through immunogenic apoptosis, necroptosis, necrosis, autophagic cell death, and pyroptosis, which exposes calreticulin and HSPs to the cell surface and/or releases ATP, HMGB1, uric acid, and other DAMPs as well as PAMPs as danger signals, along with tumor-associated antigens (TAAs), to activate dendritic cells and elicit effective antitumor immunity [ 114 , 115 ]. However, it was recently shown that dendritic cell-mediated anti-tumor immunity is compromised by cancer cells undergoing ferroptosis [ 116 ]. To respond to the tumor antigens, antigen-presenting cells (APCs) catch TAAs and neoantigens released by tumor cells, and then activate tumor-specific T lymphocytes [ 117 , 118 ]. On the surface of tumor cells infected with OVs, there are virus-specific antigens, which help in their elimination by antiviral T lymphocytes [ 119 ]. As a result, even if the virus does not reproduce well, OVs can trigger an antitumor immune response [ 106 ]. Additionally, the breakdown of the tumor's immunological tolerance is acknowledged as a major feature of OV's method of action and can destroy the tumor [ 120 , 121 ].

Additionally, the OV will infect tumor cells and control protein production, increasing viral macromolecule creation while also stimulating the expression and detection of danger signals. Indeed, these are the results of some signaling pathways that end in the release of DAMPs such as heat shock proteins (HSPs), calreticulin, uric acid, and ATP and cytokines including, interferons (IFNs), tumor necrosis factor-α (TNF-α) and IL-12, all of which help to improve immune responses [ 105 , 106 , 122 , 123 ]. Pathogen-associated molecular patterns (PAMPs), such as nucleic acids, proteins, and viral capsid elements, are also released as a result of virus-induced tumor cell death [ 124 , 125 , 126 , 127 ]. These compounds help counteract the immunosuppressive condition of the TME by promoting the migration and activation of macrophages, NK, DC, and tumor-specific cytotoxic T cells [ 128 , 129 , 130 ]. The TME is made up of tumor cells, local or penetrated non-transformed cells (e.g., cancer-associated fibroblasts, vascular endothelial cells, immune recruitment cells), secretory substances, and the extracellular matrix. In fact, this microenvironment is generally immunosuppressive and tumors produce soluble immunosuppressive agents such as nitric oxide and cytokines including IL-10 and TGF-β, resulting in active suppression of efficient anti-tumoral immune response [ 106 , 131 , 132 ]. Furthermore, Tregs and myeloid-derived suppressor cells (MDSCs) are directed to the TME, where they use the acquired immune response pathway's potential to detect and eliminate tumor cells [ 106 , 132 , 133 ].

The immunostimulatory versus immunosuppressive behavior of the TME is controlled by cytokines and immune cells [ 107 ]. OVs enable proinflammatory cytokines to enter the TME, establishing a suitable condition for DC activation. By activating antigen presentation pathways in tumors, OVs stimulate DCs to detect tumor antigens [ 134 , 135 ]. In this environment, OVs can outperform various evasion methods. For example, by treating with oncolytic reovirus, an ovarian cancer cell line expressed more MHC class I and other molecules related to antigen processing, such as the transporter associated with antigen processing (TAP) and β2-microglobulin (β2M). This action enhanced DC maturation, which resulted in adaptive immunological responses driven by CD8 T cells [ 136 ]. Moreover, in a murine model treated with an engineered adenovirus, many splenic CD11c CD8 DCs were found an tumor-infiltrating plasmacytoid DCs revealed a mature phenotype capable of priming tumor-specific cytotoxic T cell activities [ 137 ]. Furthermore, multiple studies have shown that other OVs, including vaccinia virus [ 138 ], measles virus [ 139 ], and HSV [ 140 ], can improve DC antigen presentation, which is commonly associated with enhanced expression of costimulatory/activation components such CD80, CD86, and MHC II.

The tumor-associated macrophage population is an important modulator of the immunostimulatory versus immunosuppressive behavior of the TME. Anti-viral and anti-cancer responses are related to M1 pro-inflammatory macrophages, whereas metastasis, angiogenesis, and inhibition of anti-cancer and anti-viral responses are related to M2 immunosuppressive macrophages [ 141 , 142 , 143 ]. As shown by oncolytic paramyxovirus infection of macrophages, OVs act as potent immunological triggers and are useful to modify the phenotypic activities of macrophages [ 144 ]. OVs are able to provide an inflammatory environment that encourages macrophage infiltration and activation. For instance, in a xenograft colorectal cancer model, it was shown that treatment with the oncolytic vaccinia virus GLV-1h68 caused a considerable increase in proinflammatory cytokines like IL-3, IL-6, IFN-, and CXCL10. It causes boosting the recruitment of proinflammatory macrophages to the tumor site [ 145 ]. Similar to this, a triple combined treatment including oncolytic HSV boosted macrophage recruitment and M1-like polarization, which helped to eliminate glioblastoma [ 146 ].

The typical antiviral response from healthy cells, which can limit OV reproduction and disseminate directly, is one of the earliest defenses to OVs. Type I IFN is one of the key drivers of this activity (IFN-a and IFN-b) [ 141 , 142 , 147 , 148 ]. Type I IFN plays a significant part in anti-cancer responses by triggering immune cells inside the TME, including NK cells and CD8+ T cells, and pro-inflammatory cytokines, in addition to regulating the anti-viral condition [ 148 , 149 ]. Because of its regulatory impact on NK cells and CD8+ T cells, Type I IFN enhances anti-tumor immune responses. Activated NK cells release type II interferon (IFN-γ), which suppresses angiogenesis, promotes apoptosis, and stimulates the immune system (by activating MHC class II in DCs, macrophage phagocytic activity, and CD8+ T cell responses) [ 107 ]. Type I IFN can also increase MHC class I expression in DCs, as well as co-stimulatory molecules (CD40, CD86) and the Th1 polarized response [ 141 , 142 ] (Fig.  1 ). Furthermore, the capability of OV-infected tumor cells to produce type I IFN in each tumor site, as well as the potency by which certain OVs stimulate type I IFN signaling, varies significantly among OVs, and these facts all influence the OV's potential to activate the acquired immune system against viral and cancer antigens [ 142 ]. To achieve the appropriate balance between anti-viral and anti-cancer immunity, more research into the impact of each OV is required.

Mesenchymal stem cell-based delivery of oncolytic virus challenges. MSCs might stimulate angiogenesis, tumor cell proliferation, and metastasis by release large numbers of cytokines and growth factors, including VEGF, FGF-2, βFGF, PDGF, IL-8, IL-6, CXCL1, CCL5, and SDF-1. MET during metastasis tighten epithelial connections and make therapy challenging. Hypoxic circumstances have been observed to decrease viral proliferation and lytic capacity. Type I IFN hinder intra-tumoral spread of the OVs, moreover, infection with double-stranded RNA leads to PKR activation in the cell. Excessive viral replication may result in premature MSC lysis and reduce the efficiency. VEGF vascular endothelial growth factor, FGF-2 fibroblast growth factor 2, PDGF platelet-derived growth factor, IL interleukin, CXCL c-x-c motif chemokine ligand 1, CCL5 c–c motif chemokine ligand, SDF-1 stromal cell-derived factor 1, MET mesenchymal-to-epithelial transitions, IFN I type I interferon, PKR RNA-dependent protein kinase, eIF2α eukaryotic initiation factor 2 α

Angiogenesis is a cancer characteristic that involves the supply of nutrients and oxygen to tumor cells to enhance tumorigenesis [ 150 , 151 ]. Several OVs have been demonstrated to have anti-angiogenic properties by inducing an acute disturbance of the tumor vasculature [ 152 , 153 , 154 ]. For instance, it has been found that the oncolytic vaccinia virus inhibits tumor angiogenesis, limits blood supply to tumor cells, and ultimately leads to hypoxia through impacting vascular cells [ 155 , 156 , 157 , 158 ]. In addition, OVs can destroy uninfected cancer cells by damaging tumor blood vessels and enhancing particular antitumor immune responses. The transgene-encoded proteins produced by modified viruses can assist oncolytic viruses in killing uninfected cancer cells [ 159 ].

MSC-based delivery of oncolytic adenovirus (oAds)

Since the oAds replication could be limited to malignant cells, these viruses are being evaluated in clinical trials for the treatment of several malignancies [ 160 , 161 , 162 ]. (Table 1 ) In fact, after the oAds replicates in tumor cells, the cell is lysed and more infectious virions are produced, infecting and lysing adjacent tumor cells and consequently inducing endogenous tumor immune responses which has therapeutic benefits [ 163 ]. It has been revealed that MSCs are finally lysed by oAd replication which avoids any negative side effects related to stem cell viability in vivo [ 59 , 164 ]. In addition, multiple investigations of individuals with ovarian cancer, melanoma, soft tissue or primary bone sarcoma, and other neoplasms have revealed that oAds have a high safety profile [ 10 , 160 , 161 , 162 , 165 , 166 , 167 , 168 , 169 ]. For instance, Garcia-Castro and colleagues demonstrated the safety and effectiveness of delivering numerous doses of autologous MSCs infected with ICOVIR-5, which is an optimized oncolytic adenovirus, to four children with metastatic neuroblastoma who had failed to respond to standard therapy [ 170 ]. An in vivo study also revealed that infected MSCs transport the combination of ICOVIR15 and Ad.iC9 to lung tumor sites effectively and specifically increase overall survival and tumor control [ 50 ]. Furthermore, in Stoff Khalili et al. research, hMSCs were utilized as intermediate vehicles for conditional replication oncolytic adenoviruses (CRAds) to target breast cancer and reduced the development of pulmonary metastasis, most likely due to viral replication in the hMSCs [ 90 ]. Pulmonary metastasis in patients with breast cancer is common characteristic [ 171 , 172 , 173 ] and is associated with lethal complications in these patients [ 174 , 175 ]. Furthermore, when CRAd-loaded MSC was given intravenously to mice with solid ovarian cancer, it had a considerably stronger anticancer impact and a prolonged survival time than CRAd delivered directly [ 89 ]. Moreover, it has been shown that hepatocellular carcinoma (HCC)-targeted oAd can be loaded into MSCs successfully by viral capsid modification, and oAds induce significant cancer-specific death pathways through active viral reproduction in the MSC driver. Indeed, oAd-loaded MSCs improve both oAd and MSC safety features by reducing oAd hepatic sequestration and hepatotoxicity while improving MSC clearance through viral proliferation [ 59 ]. Also, Rincon et al. revealed that MSCs carrying the oncolytic adenovirus ICOVIR5 were therapeutically effective in treating lung cancer in mice by inhibiting tumor development and encouraging T cell migration to the tumors [ 176 ]. Similar to this, MSCs infected with the oncolytic adenovirus CRAd5/F11 prevented tumor growth in a colorectal cancer subcutaneous mouse xenograft model [ 177 ]. In the other study Kaczorowski et al. [ 178 ] removed the antiapoptotic gene E1B19K from oAd and replaced it with the cell death ligand TRAIL gene. After intravenous injection of infected MSCs, adenoviral capsid protein was found in tumor xenograft tissue but not in healthy tissue, indicating tumor-specific migration [ 178 ]. Similarly, direct in vivo therapy was associated with a significantly decreased tumor size, lower Ki67 and CD24 expression, and increased caspase activation [ 160 ]. Chastkofsky et al. evaluated the potential of MSC for OV delivery for the treatment of diffuse intrinsic pontine glioma (DIPG. They utilized the survivin promoter for conditional replication of OV. Their results revealed that cells and tumors have increased expression of survivin and cell surface proteins which provide effective OV entry and replication in DIPG cells and result in more prolonged survival [ 179 ]. To elaborate, survivin is an anti-apoptotic factor that facilitates the viability and survival of cells and has been identified as a target in the treatment of several autoimmune diseases and cancers [ 180 , 181 ].

In a mouse model of breast cancer with pulmonary metastases, Zhang et al. investigated the therapeutic effects of MSCs infused with OAd expressing decorin, a naturally occurring inhibitor of TGF-β signaling. They demonstrated that MSCs boosted the therapeutic benefits of oncolytic adenoviral administration and dissemination in tumor tissues [ 182 ]. Since the oncolytic adenovirus replicates in MSCs, it is essential to balance MSC viability with viral load in order to get the best therapeutic outcome. Recently, Zhang et al. developed an all-in-one Tet-on system that capable of controlling the reproduction of OAd. The new OAd expressing Endostatin and/or IL-24 was then introduced into hUCB-MSCs for the treatment of glioma. Their findings showed that this new OAd was capable of killing glioma cells with high efficiency while sparing healthy cells [ 183 ].

Interestingly, Barlabé et al. equipped the OAdv with a therapeutic transgene to provide the strongest anticancer effects. They demonstrated that, when menstrual blood-derived mesenchymal stem cells (MenSCs) are combined with ICOVIR15-cBiTE, an OAdv producing an EGFR-targeting bispecific T-cell engager (cBiTE), the antitumor effectiveness is increased in comparison to MenSCs loaded with the unarmed virus ICOVIR15 [ 194 ]. Combining MSC-delivered OV with prodrug activation is another strategy to achieve optimum effectiveness. Under these circumstances, MSCs might convert the simultaneously delivered prodrug into cytotoxic metabolites, causing oncolysis and inhibiting tumor development without being hazardous to the host's essential organs [ 195 ].

Adoptive cell treatments for solid tumors have a significant challenge due to the immunosuppressive TME [ 196 ]. Oncolytic immunotherapy using modified OAd by infecting tumor cells, may disrupt the TME [ 197 ]. It was recently revealed that lung cancers could be successfully treated in animal models using a combination of cell carrier-delivered OAd and chimeric antigen receptor (CAR-T) cells. A binary vector including an OAd and a helper-dependent Ad (HDAd; combinatorial Ad vector (Cad)) that expresses checkpoint PD-L1 blocker and IL-12 has been demonstrated by this work to be systemically delivered by MSCs [ 198 ]. The immune checkpoint ligand PD-L1 has been found to be overexpressed in a variety of solid tumors [ 199 ]. PD-L1 binds to its receptor PD-1, and blocking it has shown therapeutic potential in cancer therapy [ 200 ]. However, when PD-L1 blockade is combined with optimal dose IL-12 delivery, it induces a synergistic effect of enhancing anti-tumor immunity in cancer patients [ 201 ].

MSC-based delivery of oncolytic herpes simplex virus (oHSV)

The oHSV has been evaluated extensively in combination with MSCs and has demonstrated promising outcomes in the treatment of gliomas, metastatic melanomas, breast, and ovarian cancers, whether administered systemic [ 49 , 184 ] or locally [ 185 ]. For example, in order to investigate efficiency of oHSV in immune-deficient and immune-competent murine models of melanoma brain metastasis, Du et al. utilized MSCs as cellular vehicles for oHSV and revealed that MSC-oHSV effectively detects metastatic tumor deposits in the brain, inhibits brain tumor development, and extends survival [ 184 ]. Furthermore, in a clinically relevant glioma model, Duebgen M, et al. infused hMSCs with oHSV and revealed that they successfully generated oHSV progeny, cause tumor resection and resistance and extended average lifespan in mice [ 185 ]. Indeed, one of the most important alternatives for GBM treatment is the oHSV, which is a naturally neurotrophic factor [ 8 , 202 , 203 ]. In addition, it has been shown that MSCs from various origins can be infected and loaded with a HER2-retargeted oncolytic HSV and the metastatic burden in the brain was shown to be reduced by more than half in NSG mice, which showed significant suppression of breast cancer brain metastases [ 49 ] (Table 1 ).

MSC-based delivery of oncolytic measles virus (oMV)

The oMV exhibits significant anticancer potential and is being studied as an innovative tumor therapy in a number of Phase I clinical studies [ 204 , 205 ]. Indeed, oMV reproduction, protein expression, syncytia formation, and oncolysis have been reported in vivo, in a variety of human tumor xenografts, such as hematologic malignancies like lymphoma [ 206 , 207 ] and myeloma [ 208 ], as well as solid tumors including ovarian cancer [ 209 ], glioblastoma [ 210 ], hepatocellular carcinoma [ 211 ], prostate cancer [ 212 , 213 ], breast cancer [ 214 ], cervical cancer [ 215 ], and gastric cancer [ 216 , 217 ]. On the other hand, MSCs have been demonstrated to be efficient carriers of attenuated oMV to ovarian tumors [ 94 ] and HCC [ 95 ]. For instance, in the hepatocellular carcinoma and murine model, two groups investigated intravenous single delivery of oMV-loaded BM-MSCs. Both measles antibody-naive and passively-immunized SCID mice showed a considerable decrease in tumor development when treated with oMV-infected BM-hMSCs [ 95 ]. Moreover, in an orthotopic ovarian cancer therapy model, treated mice with AD-MSCs as drivers of oMV survived longer in comparison to mice treated with a naked virus or uninfected MSC [ 53 ]. In addition, in a xenograft model, BM-hMSCs were shown to successfully delivering OMVs to precursor B-lineage acute lymphoblastic leukaemia (ALL) cells. Ex vivo loading of oMV into BM-MSCs was effective and oMV was replicated intracellularly without toxicity [ 186 ].

Early research [ 208 , 210 , 212 , 218 , 219 , 220 ] reported an increase in apoptotic markers in infected cells, whereas Lampe et al. demonstrated viral cells die even when apoptosis inhibitors are used, indicating that alternative cell death pathways are involved [ 221 ]. Donnelly, OG et al. revealed that Measles virus causes immunogenic cell death (ICD) in human melanoma[ 222 ].

MSC-based delivery of oncolytic myxoma virus (MYXY)

In order to cure a malignant brain tumor in mice, human adipose-derived MSCs (AD-MSCs) have been coupled with MYXV, which expresses the reporter gene green fluorescent protein (GFP) [ 187 ]. This study established the potential of AD-MSCs to produce new viral particles as well as the ability of infected cells to adhere to tumors when administered intravenously. As a consequence, mice treated with the OV-loaded MSCs had a much higher survival rate than mice treated with MSCs alone.

BM-MSCs have also shown to be permissive to MYXV replication [ 188 ]. Furthermore, the scientists showed improved antitumor activity in an immunological competent lung melanoma model following intravenous treatment, in comparison to MYXV monotherapy, employing an IL-15-armed MYXV to infect BM-MSCs. Only animals treated with the virus without cell carriers showed an enhanced proportion of circulating NK cells, suggesting that MSCs may have inhibited the immune system from recognizing the infection. Finally, tumors from animals treated with MYXV-IL-15-loaded MSCs showed an increase in pro-inflammatory cytokines, programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1), and infiltration of effector T cells, suggesting a potential antitumor immune response as a result of the therapy.

In a separate investigation, recombinant MYXV, which encodes murine LIGHT, also known as tumor necrosis factor ligand superfamily member 14 (TNFSF14), was used to infect AD-MSCs ex vivo. Results from this study showed that mice with orthotopically induced pancreatic ductal adenocarcinoma (PDAC) had increased trafficking into the pancreas compared to tumor-free animals, which led to the extended survival of the treated PDAC-seeded animals and the increased expression of important adaptive immune response markers. Administered IP and pre-loaded ADSCs with transgene-armed MYXV really enable more efficient oncolytic virus ferrying to PDAC sites and promote better tumor regression [ 189 ]. Additionally, it has been shown that combining the pre-loaded LIGHT (TNFSF14)-Armed Myxoma virus with Gemcitabine (as an antimetabolite) may be a potential strategy to enhance the therapeutic benefits of vMyx-LIGHT/ADSCs against PDAC in vivo [ 190 ] (Table 1 ).

MSC-based delivery of oncolytic Reovirus

A wide range of human malignancies, including acute myeloid leukemia, have been treated with reovirus, a naturally occurring OV [ 223 ]. The potential use of an oncolytic reovirus created by Reolysin®, (pelareorep; wild-type reovirus; Serotype 3 Dearing; Oncolytics Biotech Inc.), for the treatment of various tumor cells was examined in several clinical trials [ 224 ]. Reolysin®, a novel systemically administered promising anti-cancer drug for pancreatic, ovarian, and malignant glioma tumors, received FDA approval in 2015 [ 225 , 226 ]. The oncolytic reovirus's anti-cancer effect against the glioblastoma multiforme (GBM) cell line may be enhanced by AD-MSCs, which are a vulnerable host for the virus [ 227 ]. In vitro cancer treatment using MSCs that have been infected with reovirus type-3 Dearing (T3D) has also been examined. According to the study's findings, reovirus-infected AD-MSCs in TC-1 cells produce more NO and TNF-α than normal while producing less TGF-β1 and IL-10. Additionally, TC-1 cells were co-cultured with infected AD-MSCs, which greatly enhanced apoptosis when compared to the control [ 192 ].

In a different study, Babaei et al. examined the anti-tumor potential of adipose-derived mesenchymal stem cells (AD-MSCs) as a novel delivery system for the Dearing strain of reovirus (ReoT3D), which has the highest capacity to eradicate cancer cells among other strains in a murine model of colorectal cancer. They found that ReoT3D and MSCs together were more effective for therapy than ReoT3D and MSCs alone [ 191 ]. Wang et al. demonstrated that Human UC-MSCs harboring reovirus display antitumor effectiveness impacts for AML in the presence of Nabs via increasing CXCL10 production from hUC-MSCs [ 193 ] (Table 1 ).

Clinical studies using MSC-OV

Only a few clinical trials have used MSC-OV to treat cancer patients (Table 2 ). Autologous MSCs loaded with oAd (ICOVIR) or CELYVIR were employed in the first-in-human trial for the treatment of pediatric refractory metastatic neuroblastoma (NCT01844661) [ 38 ].

Another phase I/II clinical trial examined the adverse effects and optimal dosage of MSCs infected with the oncolytic measles virus that encodes the thyroidal sodium iodide symporter (MV-NIS) and how effectively it treats patients with recurrent ovarian, primary peritoneal, or fallopian tube cancer (NCT02068794). Also, another phase I trial, evaluated the optimum dose and side effects of the oncolytic adenovirus DNX-2401-loaded hBM-MSCs in treating patients with recurrent high-grade glioma through intra-arterial administration (NCT03896568). Also, the safety and efficacy parameters of AloCELYVIR in Metastatic uveal melanoma patients with hepatic metastases were examined in phase I/II clinical trial (NCT05047276) (Table 2 ).

Despite progress in our understanding of MSC-based OV delivery, there are still significant challenges ahead, which raises questions and concerns that are debatable and scientists have proposed a number of solutions to these problems, which are detailed below (Fig.  2 ).

MSCs feature as OVs carriers and mechanisms of MSC-released OVs in cancer treatment. OVs are maintained by MSCs from immune system responses. MSCs migrate to the tumor site via chemotaxis. There are two major methods by which OVs destroy tumors are direct cell death and the activation of anti-tumor immunity. Tumor cells secret and release DAMPs such as HSPs, calreticulin, uric acid, and ATP and cytokines including, IFNs, TNF-α and IL-12, and PAMPs, such as nucleic acids, proteins, and viral capsid elements as a result of OVs infection and oncolysis. These compounds help counteract the immunosuppressive condition of the TME by promoting the migration and activation of MQs, NK cells, DCs, and tumor-specific cytotoxic T cells. Normal cells antiviral response also includes type I IFN which can play a significant part in anti-cancer responses by triggering immune cells inside the TME. DAMPs damage-associated molecular patterns, HSPs heat shock proteins, ATP adenosine triphosphate, IFNs interferons, TNF-α tumor necrosis factor-α, PAMPs pathogen-associated molecular patterns, MQs macrophages, NK cells natural killer cells, DCs dendritic cells, TME tumor microenvironment

MSC tumorigenesis

The interaction of tumor cells with healthy cells and the stroma in the TME is getting important since these interactions have a role in critical stages of tumor progression including angiogenesis, immunomodulatory, metastasis, invasion, and apoptotic resistance [ 113 , 228 , 229 ]. Moreover, reports have argued that MSCs enhance or suppress tumor growth and metastasis through a variety of mechanisms, including the secretion of soluble molecules that trigger or repress innate and adaptive immune responses, activate or reduce angiogenesis, and sustain the cancer stem cell environment [ 230 , 231 , 232 ]. Indeed, MSCs can alter the rate of tumorigenesis depending on the circumstances [ 36 , 233 ]. According to studies, MSCs release trophic factors that can boost tissue angiogenesis, cell proliferation, and cell survival [ 234 , 235 , 236 ]. For example, large numbers of cytokines and growth factors that stimulate angiogenesis, including VEGF, FGF-2, βFGF, PDGF, IL-8, IL-6, angiopoietin, and TGF, are released by MSCs and contribute to the development of tumor angiogenesis [ 237 , 238 , 239 ]. Additionally, cancer-associated fibroblasts (CAFs) actively encourage tumor angiogenesis by producing chemokines and cytokines such as IL-4, IL-8, IL-6, TNF, CXCL12, TGF, and VEGF which have anti-inflammatory and pro-angiogenic properties [ 240 ]. CAFs differentiation is enhanced by interactions between MSC and tumor cells [ 237 , 241 , 242 ]. Furthermore, MSCs stimulate epithelial-mesenchymal transition (EMT) by secreting growth factors and cytokines such as HGF, PDGF, EGF, and TGF, which cause the production of transcriptional regulators of EMT like Slug, Snail, Zeb1, and Twist [ 243 , 244 ]. Evidence suggests that abnormal EMT enhances tumor metastasis, drug resistance, and tumor growth [ 245 ]. The epithelial cell-related proteins E-cadherin, ZO-1, and -catenin/plakoglobin are downregulated during the EMT while mesenchymal proteins including fibronectin, N-cadherin, smooth muscle actin, and vimentin are increased [ 71 , 246 ]. In addition through the release of chemokines such as CXCL1, CCL5, CXCL5, CXCL8, and CXCL7 by MSCs, tumor cell migration to metastatic sites is accelerated [ 247 , 248 ]. It has been also shown that MSCs release large amounts of CXCL12 (SDF-1), which control the invasion and migration of tumor cells that express CXCR4 [ 249 , 250 ]. MSCs polarization in response to substances released by the tumor, is another explanation for the conflicting results, which either forces the cells into a tumor-promoting or suppressive action. Accordingly, an in vitro co-culture of MSC1 with several cancer cell lines reduced the growth of tumors, while the MSC2 co-culture had a contradictory outcome. Similarly, MSC1 treatment of tumors established in immune-competent mice reduced tumor growth and metastasis, whereas MSC2 treatment promoted tumor development and dissemination [ 159 ]. Indeed, inhibiting or activating certain MSC TLRs might be a promising approach for enhancing the anticancer effects of OV oncolysis by adding MSC immune-stimulatory features.

As a result, a greater understanding of the particular molecular processes behind these pro-tumorigenic actions is essential for further improving anti-cancer therapeutic approaches. Enhancing the therapeutic effects for cancer patients would be achievable through the reduction of MSC recruitment into tumor areas and the suppression of their tumor-supportive actions, particularly with the combination of other therapeutic methods, such as immunotherapy.

However, while viral replication within MSCs is a desirable feature, excessive viral replication may result in premature MSC lysis and reduce the overall efficiency [ 59 ]. It has been demonstrated that a large initial viral dosage reduces total MSC survival with just a little increase in overall viral production [ 59 ].

Antiviral responses

Patients’ antiviral immune responses are a major concern, limiting the impact of oncolytic viruses [ 251 , 252 ]. The patient's antiviral defense is triggered and recruited to limit virus reproduction and dissemination, resulting in viral elimination and the treatment impact being lost [ 106 ]. Antiviral cytokines, including various forms of IFN, are a barrier to an efficient anti-tumor response to OV because hinder intra-tumoral spread of the OVs [ 253 ]. Several studies have utilized histone deacetylase (HDAC) inhibitors to promote epigenetic changes and reduce antiviral cytokine responses in the TME to resolve this challenge [ 254 , 255 , 256 ]. Furthermore, infection with double-stranded RNA leads to PKR activation in the cell. PKR can phosphorylate the α-subunit of eukaryotic initiation factor 2 α (eIF2α) which keeps it inactive and prevents viral protein production [ 257 , 258 ]. However, in vitro and in vivo studies have shown that Sunitinib inhibits the antiviral enzymes RNase L and PKR, which impedes antiviral innate immune responses [ 258 ].

OV optimizing

When determining the ideal OV treatment method, inherent qualities should be regarded. Each OV family will have its genomic complexity, replication methods, lytic qualities, transgene packaging capacity, and immune response-inducing ability to activate anti-tumor immunity. Since different OVs have distinct tumor tropisms, it has been difficult to identify precise molecular biomarkers that predict particular anti-tumor efficacy for each OV [ 133 , 259 ]. Moreover, optimizing the initial OVs loading dosage is a critical factor that improves loading efficacy and affects treatment effectiveness [ 260 ]. Additionally, the oncolytic virus lifecycle's time is an essential factor in the carrier cells' tumor-homing potential. Indeed, before the viral progeny is released, the delivery cell must concentrate in tumor sites to provide effective delivery of the therapeutic virus [ 261 ].

Targeting methods

MSCs can be targeted using a variety of methods, such as physical, physiological, and biological ones that attempt to increase their density in a specific area [ 262 ]. Physical targeting is inserting cells directly into the area that requires therapy via surgical methods or guiding techniques like catheters or external magnets [ 263 , 264 , 265 ]. In addition, a different approach is to contain therapeutic cells in a matrix or devices that maintain cells in the transplant area [ 262 ]. For example, it has been demonstrated that MSC encapsulation in a biodegradable, synthetic extracellular matrix dramatically boosted their survival in the GBM excision cavity while permitting the production of anti-cancer proteins [ 185 , 266 , 267 ]. Another method employs physiological mechanisms like the systemic circulation to transfer the cells rather than active cell-mediated migration [ 262 ]. For instance, cells frequently become caught in the lungs' capillaries. To distribute MSC-mediated treatments to the lungs, this effect can be used [ 54 , 90 ]. The MSCs trapping in the lung seems to be dependent on several factors, such as administration route and vessel size. Intravenous administration of MSCs results in an accumulation of cells in the lungs, which are then redistributed to the kidneys, spleen, and liver. Arterial injection bypasses the lungs, so MSCs are widely distributed throughout the rest of the body. There is insufficient systemic biodistribution during intramuscular, intraarticular and intradermal administration [ 268 , 269 ]. In spite of the route of injection of MSCs [ 66 ], the size of the vessels are also play a key role in MSCs trapping in lung. Scherpfer et al. showed that the average size of MSCs is larger than the size of pulmonary capillaries. Therefore, large amounts of administered MSCs can become trapped in the capillaries of the lung and prevent access to other organs. In addition, vasodilator could be reduced lung localization [ 270 ].

Furthermore, biological targeting techniques have been developed to satisfy the demand for greater target stringency following systemic administration of MSCs, particularly when the disease to be treated is extensive, as in metastases [ 271 , 272 ]. It includes evidence-based techniques aiming at enhancing MSC homing, binding selectivity to a target tissue, and persistence within the target environment [ 262 ]. Indeed, to regulate MSC homing potential, various approaches have been established, including altering the MSC culture conditions to promote the production of homing-related compounds, redesigning the cell membrane to increase homing, and adjusting the target tissue to better attract MSCs [ 273 ].

OV penetration

Epithelial junctions function as an obstacle to the intracellular infiltration of OVs, particularly adenoviruses, in carcinomas [ 274 ]. Indeed, phenotype changes during metastasis, including EMT and later mesenchymal-to-epithelial transitions (MET), which tighten epithelial connections and make therapy challenging [ 275 , 276 ]. Yumul et al. created epithelial junction openers (JO) by modifying Ad5Δ24. They found that oncolytic Ads that express JO had a substantially higher anti-tumor activity than unmodified viruses [ 274 ]. Moreover, the extracellular matrix (ECM) and cellular connections are significant barriers that are related to the spread and penetration of OVs. In fact, OVs must cross the complex ECM to reach tumor cells and lysis them [ 277 ]. Pre-treatment of cancer with collagenase [ 278 ] or co-administration of hyaluronidase with oncolytic adenoviruses [ 279 ] resulted in increased viral dissemination. Additionally, altering OVs to express matrix metalloproteinases-1 and -8 causes cancer-associated sulfated glycosaminoglycans to be degraded, resulting in improved viral dispersion and treatment efficiency [ 280 ]. Tumor cell apoptosis also promotes viral dissemination. For instance, Nagano et al. found that cytotoxic substances induced apoptosis and activated caspase-8, resulting in greater intratumoral uptake and anti-cancer effect of oncolytic HSV. They hypothesized that reducing or eliminating apoptotic tumor cells resulted in channel-like structures and empty areas, enabling oncolytic HSV to disseminate more easily [ 281 ].

Hypoxic effect

Hypoxia is a characteristic of solid tumors that emerges throughout the formation and development of the tumor and has been demonstrated to have paradoxical impacts on OVs [ 282 ]. Hypoxic circumstances have been observed to decrease viral proliferation and lytic capacity without changing the expression of surface receptors [ 283 , 284 ]. Because hypoxia may cause cell cycle arrest, this feature might influence the capability of oADs and other viruses that rely on cell cycle advancement to reproduce [ 284 ]. Clarke et al. created an oncolytic adenovirus in which the expression of the E1A gene is regulated by the hypoxia-response factor-containing promoter to counteract hypoxic suppression of viral reproduction and to get the benefits of hypoxic conditions for homing [ 285 ]. However, in 2009, two groups revealed that a hypoxic condition increases oncolytic HSV viral proliferation [ 286 , 287 ]. This might be due to HSV's intrinsic affinity to low-oxygen cells or DNA damage caused by oxygen-derived free radicals, which promotes HSV reproduction [ 286 ].

Treatment durability

Tumors frequently recur after great initial treatment results. Stem cell treatment using a single substance, like other chemotherapies, isn't always successful in removing tumors [ 288 , 289 ]. As a result, a reasonable medication combination should be determined [ 290 ]. Many different combination therapies have been tried to see whether they can help with treatment persistence. For instance, irradiating cancer cells leads them to release molecules that promote MSC penetration across integral basement membranes, resulting in an increase in the amount of MSCs in cancers [ 291 ].

Modification

In addition to their inherent potential to lyse cancer cells, OVs can be modified to improve their lytic activity. For example, adenoviruses expressing the herpes simplex virus-1 thymidine kinase (HSV-1 TK) under the osteocalcin promoter have been designed to target bone cancers. HSV-1 TK could convert thymidine analogs, such as ganciclovir into monophosphates, which stop DNA synthesis and trigger cell death By incorporating into the DNA of reproducing cells [ 292 , 293 , 294 ]. OVs have been modified to improve immune responses even further. Most transgenes are designed to induce an adaptive immune response against cancer antigens or to contribute to the treatment of immune cell-depleted malignancies. Including, cytokines, chemokines, inhibitory receptors, co-stimulatory receptors, bispecific cell engagers, immunological ligands, and combinations of any of these [ 133 , 295 , 296 ]. For instance, researchers have engineered oncolytic viruses which can express IL-2, IL-12, IL-15, IL-6, IL-21, IL-18, IL-24, and granulocyte–macrophage colony-stimulating factor (GM-CSF) and activate various aspects of the immune system [ 295 , 296 ]. Moreover, the immunosuppressive TME might be altered by inserting an immune stimulatory chemical into OV genomes. The most often utilized example is GM-CSF, which has been inserted into OV genomes as an immune stimulatory molecule to promote the maturation and recruitment of APCs, particularly DCs, as well as the recruitment of tumor antigen-specific T cells and NK cells [ 109 ]. On the other hand, one aspect of transgenic-armed OVs is that immune activation can be delayed depending on the viral promoter that regulates the transgene or by controlling protein translation. To avoid an overly-rapid immune response, the expression of transgenes should be postponed until the viral oncolysis is at its maximum [ 297 ]. Additionally, the kind of transgenic and the number of transgenes that may be included in a single viral construct are both influenced by the type of virus. Unlike DNA viruses, which can handle more transgenes without harming replication, RNA viruses generally have a shorter genome and can only encode a restricted number of them [ 133 ]. Furthermore, a modified oncolytic adenovirus expressing the TRAIL gene was recently utilized to treat a mouse model of pancreatic ductal adenocarcinoma (PDAC), a malignant and lethal malignancy with a poor prognosis and few treatment options. The study revealed that in a PDAC animal model, AD-MSCs carrying TRAIL specifically homed to the cancer site and significantly slowed tumor growth, with no toxicity or adverse effects [ 178 ].

MSCs can be also genetically manipulated or preconditioned to increase their intrinsic features, such as improved migration, adhesion, and survival, as well as reduced premature aging. For example, to improve MSC migration, CXCR1, 4, and 7 were overexpressed which CXCR1 binds to IL-8 and CXCR4 and CXCR7 bind to SDF-1. Also for increasing MSC adhesion ability, MSCs were genetically engineered to express higher levels of integrin-linked kinase (ILK) [ 298 ].

Conclusion and prospect

MSCs improve the anticancer efficacy of virotherapy in a variety of ways. Indeed, MSCs act as a reproduction site for OVs, allowing for the generation of more virions, which is advantageous for virotherapy. In addition, MSCs' tumor tropism and immunosuppressive activity enable the virus to specifically target the cancer site, increasing viral spread, and survival. MSCs, on the other hand, generate cytokines that attract immune cells to the TME, increasing the anticancer immune response. Moreover, oncolysis triggers the production of danger signal including TAAs and DAMPs/PAMPs, which stimulate local anticancer immune responses and alter the TME from immunosuppressive to immunostimulatory [ 45 , 51 ].

Integrating MSCs with more effective OVs is a reasonable move towards enhancing therapeutic outcomes. Currently, there are four ongoing clinical trials using the OV-loaded MSCs for cancer therapy, which offer up a wide range of combinations with MSCs [ 299 ].

Altogether, further development of MSCs-OVs therapies may rely on a multifaceted strategy to select design parameters to improve the safety profile and efficacy of carrier cells, improve viral replication in MSCs, and establish patient eligibility criteria. For overcoming these obstacles some efforts have performed. For example, by manipulating the MSCs, it is feasible to enhance the clinical result. Polymers or other viral capsids might potentially be used to improve infectivity and viral replication [ 300 , 301 ]. Also to control the adenovirus’s replication within MSCs, an all-in-one Tet-on system has been developed, which could help future studies to reach the optimum therapeutic effect of the oncolytic virus [ 183 ].

To summarize, although existing clinical trials will help to clarify the therapeutic efficacy of MSCs as OV cell carriers, further efforts should be undertaken to translate current viral and cellular preclinical achievements to the clinic, either as monotherapy or in combination with radiation, chemotherapy, or even immunotherapies.

Availability of data and materials

Not applicable.

Abbreviations

Oncolytic viruses

Tumor microenvironment

Mesenchymal stem cells

Umbilical cord derived MSCs

Bone marrow MSCs

Damage-associated molecular patterns

Pathogen-associated molecular patterns

Oncolytic adenovirus

Conditional replication oncolytic adenoviruses

Herpes simplex virus

Oncolytic measles virus

Epithelial-mesenchymal transition

Mesenchymal-epithelial transitions

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Ghasemi Darestani, N., Gilmanova, A.I., Al-Gazally, M.E. et al. Mesenchymal stem cell-released oncolytic virus: an innovative strategy for cancer treatment. Cell Commun Signal 21 , 43 (2023). https://doi.org/10.1186/s12964-022-01012-0

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Study documents safety, improvements from stem cell therapy after spinal cord injury

Susan Barber Lindquist

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ROCHESTER, Minn. — A Mayo Clinic study shows stem cells derived from patients' own fat are safe and may improve sensation and movement after traumatic spinal cord injuries . The findings from the phase 1 clinical trial appear in Nature Communications . The results of this early research offer insights on the potential of cell therapy for people living with spinal cord injuries and paralysis for whom options to improve function are extremely limited.

In the study of 10 adults, the research team noted seven participants demonstrated improvements based on the American Spinal Injury Association (ASIA) Impairment Scale. Improvements included increased sensation when tested with pinprick and light touch, increased strength in muscle motor groups, and recovery of voluntary anal contraction, which aids in bowel function. The scale has five levels, ranging from complete loss of function to normal function. The seven participants who improved each moved up at least one level on the ASIA scale. Three patients in the study had no response, meaning they did not improve but did not get worse.

"This study documents the safety and potential benefit of stem cells and regenerative medicine," says Mohamad Bydon, M.D. , a Mayo Clinic neurosurgeon and first author of the study. "Spinal cord injury is a complex condition. Future research may show whether stem cells in combination with other therapies could be part of a new paradigm of treatment to improve outcomes for patients."

No serious adverse events were reported after stem cell treatment. The most commonly reported side effects were headache and musculoskeletal pain that resolved with over-the-counter treatment.

In addition to evaluating safety, this phase 1 clinical trial had a secondary outcome of assessing changes in motor and sensory function. The authors note that motor and sensory results are to be interpreted with caution given limits of phase 1 trials. Additional research is underway among a larger group of participants to further assess risks and benefits.

The full data on the 10 patients follows a 2019 case report that highlighted the experience of the first study participant who demonstrated significant improvement in motor and sensory function.

Watch: Dr. Mohamad Bydon discusses improvements in research study

Journalists: Broadcast-quality sound bites are available in the downloads at the end of the post. Please courtesy: "Mayo Clinic News Network." Name super/CG: Mohamad Bydon, M.D./Neurosurgery/Mayo Clinic.

Stem cells' mechanism of action not fully understood

In the multidisciplinary clinical trial, participants had spinal cord injuries from motor vehicle accidents, falls and other causes. Six had neck injuries; four had back injuries. Participants ranged in age from 18 to 65.

Participants' stem cells were collected by taking a small amount of fat from a 1- to 2-inch incision in the abdomen or thigh. Over four weeks, the cells were expanded in the laboratory to 100 million cells and then injected into the patients' lumbar spine in the lower back. Over two years, each study participant was evaluated at Mayo Clinic 10 times.

Although it is understood that stem cells move toward areas of inflammation — in this case the location of the spinal cord injury — the cells' mechanism of interacting with the spinal cord is not fully understood, Dr. Bydon says. As part of the study, researchers analyzed changes in participants' MRIs and cerebrospinal fluid as well as in responses to pain, pressure and other sensation. The investigators are looking for clues to identify injury processes at a cellular level and avenues for potential regeneration and healing.

The spinal cord has limited ability to repair its cells or make new ones. Patients typically experience most of their recovery in the first six to 12 months after injuries occur. Improvement generally stops 12 to 24 months after injury. In the study, one patient with a cervical spine injury of the neck received stem cells 22 months after injury and improved one level on the ASIA scale after treatment.

Two of three patients with complete injuries of the thoracic spine — meaning they had no feeling or movement below their injury between the base of the neck and mid-back — moved up two ASIA levels after treatment. Each regained some sensation and some control of movement below the level of injury. Based on researchers' understanding of traumatic thoracic spinal cord injury, only 5% of people with a complete injury would be expected to regain any feeling or movement.

"In spinal cord injury, even a mild improvement can make a significant difference in that patient's quality of life," Dr. Bydon says.

Research continues into stem cells for spinal cord injuries

Stem cells are used mainly in research in the U.S., and fat-derived stem cell treatment for spinal cord injury is considered experimental by the Food and Drug Administration.

Between 250,000 and 500,000 people worldwide suffer a spinal cord injury each year, according to the  World Health Organization .

An important next step is assessing the effectiveness of stem cell therapies and subsets of patients who would most benefit, Dr. Bydon says. Research is continuing with a larger, controlled trial that randomly assigns patients to receive either the stem cell treatment or a placebo without stem cells.

"For years, treatment of spinal cord injury has been limited to supportive care, more specifically stabilization surgery and physical therapy," Dr. Bydon says. "Many historical textbooks state that this condition does not improve. In recent years, we have seen findings from the medical and scientific community that challenge prior assumptions. This research is a step forward toward the ultimate goal of improving treatments for patients."

Dr. Bydon is the Charles B. and Ann L. Johnson Professor of Neurosurgery. This research was made possible with support from Leonard A. Lauder, C and A Johnson Family Foundation, The Park Foundation, Sanger Family Foundation, Eileen R.B. and Steve D. Scheel, Schultz Family Foundation, and other generous Mayo Clinic benefactors. The research is funded in part by a Mayo Clinic Transform the Practice grant.

Review the study for a complete list of authors and funding.

About Mayo Clinic Mayo Clinic is a nonprofit organization committed to innovation in clinical practice, education and research, and providing compassion, expertise and answers to everyone who needs healing. Visit the  Mayo Clinic News Network  for additional Mayo Clinic news.

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• Published: 04 July 2022

Female germline stem cells: aging and anti-aging

• Wenli Hong 1 , 2 ,
• Baofeng Wang 3 ,
• Yasha Zhu 1 ,
• Jun’e Wu 1 ,
• Shuyi Ling 1 ,
• Ziqiong Zhou 1 ,
• Yuqing Dai 1 ,
• Zhisheng Zhong 1 &
• Yuehui Zheng 1  

Journal of Ovarian Research volume  15 , Article number:  79 ( 2022 ) Cite this article

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The delay of ovarian aging and the fertility preservation of cancer patients are the eternal themes in the field of reproductive medicine. Acting as the pacemaker of female physiological aging, ovary is also considered as the principle player of cancer, cardiovascular diseases, cerebrovascular diseases, neurodegenerative diseases and etc. However, its aging mechanism and preventive measures are still unclear. Some researchers attempt to activate endogenous ovarian female germline stem cells (FGSCs) to restore ovarian function, as the most promising approach. FGSCs are stem cells in the adult ovaries that can be infinitely self-renewing and have the potential of committed differention. This review aims to elucidate FGSCs aging mechanism from multiple perspectives such as niches, immune disorder, chronic inflammation and oxidative stress. Therefore, the rebuilding nichs of FGSCs, regulation of immune dysfunction, anti-inflammation and oxidative stress remission are expected to restore or replenish FGSCs, ultimately to delay ovarian aging.

Introduction

Because populations around the world are aging fast, the issue of aging has become a growing concern. With aging, the body's immunity and regenerative response to damage decreases, leading to development of aging-related diseases. One of the underlying causes of senescence-induced functional decline is thought to be the depletion of in situ stem cell function. Changes in the overall environment, ecological niche, and stem cells themselves related to aging contribute to this decline. Therefore, restoring stem cell function at the cellular level may result in individual rejuvenation at the organismal level, which could shed light on the development of new solutions to age-related dysfunctions and diseases [ 1 ]. With the delay of the age of first childbearing and the population structure in the modern society, the ovarian aging and related problems are becoming more serious. Ovaries cease obviously earlier than the rest organs of the body [ 2 ], which in turn ushers the start aging of whole body. Along with the substantial extension of woman life span in modern society, the asynchronous aging of ovary and whole body becames a sharp conflict with pursueing better physical and psychological well-being in woman at advanced-age. At the contemporary era in which infertility is regareded as one of the three highest diseases in humans, infertility rates soar as high as 15–20% in developed countries. In China, infertility rate has increased from 3% to 15–18% in the past four dacades, of which about 40% is related to ovarian aging in female patients. In the whole population of childbearing age women, 1–3% of women reach pathological menopause, or pathological ovary function decline, also called premature ovarian failure (POF), before the age of 40 [ 3 ]. Due to longer life span and increasing infertility incidence, ovarian aging become a huge challenges to woman health of modern society. Therefore, delaying ovarian aging is a hot key node of preserving fertility of advanced age women, POF patients or cancer patients, improving women's health and quality of life, as well as coping with aging population structure!

FGSCs aging and ovarian aging

The underlying mechanisms for the aging of the ovary are still poorly understood, partically because it is a complex biological process in which many factors interact internally and externally. Compared with the “evergreen” male testeis, female ovaries in advanced age women are more like “rotten root of old tree”. What makes this big different? The researchers believe that the FGSCs aging directly determines the ovarian aging. In phyicilogical conditions, when women reach their advanced age, the stem cells in their ovaries are exhausted, they face menopause and symptoms of hypoestrogene, while male enjoy their old age life without dramatic decline of their testes function. They still have the ability to father as long as their spouses are young enough.

Evidence of FSCG's existence in ovaries

Adult stem cells are undifferentiated cells that exist in an adult individual and are present in various tissues and organs of the body. They can be infinitely self-renewing and have the potential of targeted differentiation in an organism, which not only play an important role in the organogenesis, but also have an amazing ability to maintain and sustain tissue organ regeneration [ 4 ]. The presence of testicular stem cells was first described over 40 years ago, and this is the source of the adult's continued sperm production and maintanance of "evengreen" testicular function. Whether mammal's ovaries have FGSCs to supplement the original follicle pool after birth has been debated for nearly one century. The earliest report to answer this debate dates back to the late nineteenth century by German autopsist Wilhelm Waldeyer, who was also famous for creating the concepts of “chromosome” and “neuron”. He hypothesized that adult female mammals lost the de novo oocyte renewal abilitybased on their work mainly of histological study that showed the absence of excess oocyte production on post-perinatal period. The hypothesis dominated the academic circles for long time [ 5 ]. In the early 1950s, Zuckerman declared that there was no proof that females formed new oocytes after birth, adhering the concept of a restricted (stationary) mammalian ovarian reserve had been the main dogma of the cycle [ 6 ]. Unstill recent years, more and more researchers reported that the ovarian surface epithelium contains germ cells that form follicles. The fate of such germ cells is full of uncertainty, since many of them are either lost during migration or undergo cell death [ 7 , 8 ]. In 2004, Johnson discovered the consumption rate of non-atretic follicles was less than the atresia follicle forming rate in mice. Hence, they believe there are renew of follicles in mice and come up with FGSCs theory, which was a big challenge to ovarian follicle fixed theory [ 9 ]. Later, scientists isolate cells that could be subcultured in vitro and express the both stem and germ cell-specific protein markers in mice, adult mice, rats, and human ovarian tissue cortex respectively. Using a variety of methods, including stem cell culture and expansion, stem cell transplantation, genetic modification and gene editing, in vivo cell lineal tracking, the researchers confirmed existence of FGSCs in the postnatal ovaries in a variety of mammals, including humans, even old women ovarian surface epithelium, and observed that FGSCs had the ability to direct differentiation into eggs, continuously replenish follicle pools, and restore progeny to infertile model. FGSCs with GFP were transplanted into infertility mice, and both mature follicles with GFP and offspring with GFP were is covered obtained, which provided the most direct evidence of FGSCs existence [ 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 ]. Our team also successfully isolated FGSCs from the ovarian surface epithelium of mice. In our study, FGSCs could be stably passed on by generation, and could increase the infertile mice follicles number and successfully produce offspring after transplantation [ 31 , 32 , 33 ]. Having features of embryonic stem cells, OSE cell layer is considered as the "germ epithelium", in which it serves as the bipolar progenitor cell of oocytes and granulosa cells. In order to pinpoint the exact propotion of FGSCs in ovary, White's team evaluated that the population of FGSCs in mouse ovaries represented 0.014% ± 0.002% of the total cell population [ 17 ]. FGSCs expressed markers of both pluripotency stem cells and germ cells, such as Ddx4(VASA), Dppa3 (major maternal effect gene maintaining pluripotency), Prdm1 (early germ cells marker as transcriptional repressor), Pou5f1(POU Class 5 Homeobox 1), Dazl, Ifitm3, but not Nanog (pluripotency marker), Fig. l a, Kit, Sycp3 and Zp3 [ 24 ]. Afer injecting adult FGSCs into fetal ovaries, Sharma found that foreign stem cell participatied various events during oogenesis and follicle assembly. In the intricate expriment, author confrimed the exitence of stem cell in adult ovaries and have the potential to form follice and oogenesis [ 34 ].

Classification of FGSCs

FGSCs’ sizes vary considerably, ranging from 2–8 um [ 12 , 18 ]. Several research groups reported the existence of two stem cell populations (VSELs and OSCs) in mice, rabbits, sheep, marmosets and human ovaries [ 19 , 35 , 36 ]. Several years later, Esmaeilian's group confirmed VSELs have the capacity to be differentiated into oocyte-like structures [ 37 ]. Evidence began to accumulate that two types of stem cells, resting stem cells and activated stem cells, exist in various organs of the adult body [ 38 , 39 ]. Likewise, the two stem cell populations consist of relatively quiescent very small embryonic-like stem cells (VSELs) and their direct progeny, the "progenitor cells", called FGSCs. Being relatively quiescent, VSELs experience asymmetrical cell divisions to become progenitor cells that divide rapidly, expanding clonal offsprings through symmetrical cell divisions. These expanded progenitor cells form cysts, and ultimately differentiate into oocytes in the ovaries [ 35 , 36 , 37 , 40 , 41 , 42 , 43 , 44 , 45 ]. Such stem/progenitor cells express follicle-stimulating hormone (FSH) receptors and are activated by FSH. VSELs maintain life-long homeostasis, and may survive radiotherapy and chemotherapy. Its impairing lead to host age-related senescence due to loss of function caused by impaired ecology, and the presence of overlapping pluripotency markers suggests that they may also be associated with epithelial ovarian cancer [ 46 , 47 , 48 , 49 , 50 ].

FGSCs and remodeling of ovarian function

FGSCs were defined as committed progenitors, capable of renewing and differentiating into oocytes and remodeling of ovarian function. In 2009, Virant-Klun’s team isolated and obtained FGSCs in postmenopausal women, after induced differentiation in vitro, were detected to express zona pellucida 2 (ZP 2 ),observed to have a polar—like structure, shaped like an oocyte [ 12 ]. In the same year, Zou and colleagues identified and isolated FGSCs in neonatal and adult mouse ovaries and cultured them in vitro. They found that they had the potential to proliferate and were passed on for over 60 generations, and successfully established mouse FGSCs lines. FGSCs, transfected with GFP, were transplanted into the ovaries of infertility model mice. Transplanted cells underwent oogenesis and the mice produced offspring that had the GFP transgene. These findings contribute to basic research into oogenesis and stem cell self-renewal and open up new possibilities for applications of FGSCs in biotechnology and medicine [ 13 ]. White’s team isolated and purified female germline stem cells from adult mouse ovaries using the FACS. They also isolated FGSCs from the ovaries of adult women, and transplanted them into the cortical layer of mouse ovaries after in vitro culture. Immature oocytes were found in the mouse ovaries, and ovarian granular cells were formed around the ovaries [ 17 ]. In 2012, Hu and his colleagues also separation FGSCs from mice, and added GSK3 inhibitor-Bio into the culture system to make FGSCs induced differentiation to oocytes. PCR and immunohistochemical results showed that stem cell markers Oct4 expression was decreased and oocyte markers were increased [ 16 ]. However, are active FGSCs available in postnatal mouse ovaries? By labeling small germ cells expressing Oct4, Guo's team tracked their fate for up to 4 months and observed for the first time the presence of FGSCs with active function in adult mouse ovaries [ 23 ]. In addition, Satirapod’s team reported that mouse FGSCs express E 2 receptor-α (ERα). During oogenesis process, ERα has interaction with the E 2 stimulated by retinoicacid gene 8 (Stra8) promoter, which can drive Stra8 expression [ 30 ]. These discoveries establish a critical physiological role for FGSCs in oogenesis. However, whether FGSCs can develop into functional oocytes in vitro and its characteristics have not been reported. Wu and her team used the 3D culture system to establish the ovarian organoid model derived from mouse FGSCs. Like a normal ovary, the ovaries are filled with follicles and secrete hormones. Through single-cell sequencing, Wu's team revealed that the ovarian organoids contained 7 cell groups, including germ cells, granulosa cells, follicular membrane cells and fibroblasts. Follicles isolated from ovarian organoids can develop into mature oocytes by in vitro culture. Mature oocytes can produce normal offspring by in vitro fertilization [ 51 ].

Accumalating evidence suggest FGSCs transplantation can prolongs fertility age, cure infertility, and recover ovarian function of cancer patients. Being evently matched function of primordial germ cells, ovarian pluripotential VSELs can be differentiated into an oocyte-like structure which may bypass many obstacles that ES/iPS cells therapy faced.

Factors for FGSCs aging

Niche and fgscs aging.

Schofield presented stem cell niche hypothesis in 1978 [ 52 ]. The niche is the microenvironment for stem cells relay on and dwell. In gerenal, the niche is composed of extracellular matrix, niche cells, granulocytes, blood vessels, immune cells and secreted factors. According to the conventional view, stem cell aging leads to the senescence of organs. Its components are complicated and Its functional state can decide the subsistence of stem cells.

In the gonads of drosophila, the FGSCs niche could produce bone morphogenetic proteins (BMP), which act as ligands to FGSCs receptors and increase the expression level of BMP by inducing BMP signal cascade amplification [ 53 ]. Besides, the escort cells in the drosophila germinal stem cell niche directly affect FGSCs via GTPaseRho regulation and functional defect of Rho increase abnormal BMP level in the niche, leading to accumulation of undifferentiated single germ cells [ 54 ]. In consideration of that FGSCs niche structure and function in mammals are less clear than that of drosophila, we believe that the mammal niche is similar to the drosophila niche. The niche of ovaries in mammals maybe includes follicular membrane-stromal cells, granulosa cells, extracellular matrix, blood vessels, immune system-related cells and cytokines [ 55 ]. Bukovsky observed that niche of FGSCs formed during early embryonic development consist of nonspecific ovarian monocyte-derived cells(MDCs), T cells, and vascular endothelial cells, whereas nests of adult ovarian germinal stem cells consist of primary CD14 + MDCs, activated HLA-DR + MDCs and T cells [ 56 ]. Furthermore, after transplantation of ovarian tissue from senescent mice into the ovaries of young mice, the good status of donor primordial follicles with GFP were observed in the host mice; However, after transplanting ovarian tissue from young mice into older mice, ovarian tissue of the young mice were found to have reduced number of follicles and lack of mature follicles [ 57 ]. Recently, after transplantating stem cell from old mouse ovaries into young mouse ovary ovaries,Sharma found exitency of stem cell and differentiate, but fail to form follicle, which hint that we still know little about niche and the interaction between niche and stem cells [ 58 ]. Bhartiya and her team reported a series excellent expriments demonstrated that very small embryonic-like stem cells (VSELs), being dorment in most time, can survive oncotherapy, spontaneously differentiate into gametes after tranplantation, showing big potential to regenerate de novo functional gonads in the fufure [ 40 , 44 , 47 ]. The findings also imply that stem cell(VSELs)may altert niche’s habitability by some mechism so far we don’t know yet. Similiar to pluripotent stem cells, very small embryonic-like stem cells (VSELs) in adult gonads, developmentally equivalent to migratory primordial germ cells, can survives oncotherapy due to their quiescent nature [ 44 , 59 , 60 , 61 ]. Unfortunately, the stem cell's residence– niche was impaired by oncotherapy [ 57 , 62 , 63 , 64 ]. Transplanting niche cells (mainly refer to Sertoli or mesenchymal cells) can regenerate the non-functional gonads [ 65 , 66 ]. This approach has esulted in the birth of fertile offspring in mice. Besides it's safty, this approach and strategy show a strong application prospect. It could be used as first line treatment for permanent restoring gonadal function in POF and cancer patients.

Accumulating evidence suggest FGSCs niche is the key link to ovarian failure. FGSCs niche might be more important than aging of FGSCs themselves.

Immune system,inflammatory factors and FGSCs aging

The immune system, composed of immune organs, immune cells and secreted factors, is essential for the body to defend itself against external damage. The immune system can be classified into innate and adaptive immunity. Adaptive immunity can be further categorized into cell-mediated immunity and humoral immunity. The immune system has three important functions.(1) immune defense function, which fights against the invasion of pathogenic microorganisms such as viruses, bacteria, andfungus; (2) immune homeostasis function, which removes aging and dead cells to maintain the body's juvenscence; (3) Immune surveillance functions, or in more understandable term, identifying, killing and eliminating mutated cells to prevent cancer. These functions are key to maintaining good health, while dysfunctional immune function contributes to disease. Beside the role in maintaining body homeostasis, the immune system also has a crucial role in regulating ovarian function. The immune system coordinates the development of the ovaries, follicle maturation, and ovulation. As early as 1970s, researchers devoted considerable effort to understanding the relationship between the ovary and the immune system. Sakakura and Nishizuka observed that the ovaries of thymus-free mice fail to mature after birth [ 67 ]. Russell et al. noted that the thymocytes of wild-type female mice can inhibit cyclophosphamide and X-ray induced superovulation [ 68 , 69 ]. As early as 1979, Bukovsky and Presl proposed a hypothesis that the immune system is an important player in ovarian function [ 70 ]. Since then, this hypothesis has been supported by a growing number of studies. For example, thymosin injection in neonatal nude mice promotes maintenance of follicle-stimulating hormone and luteinizing hormone levels during later reproductive maturation, rescuing abnormal development of the ovary in nude mice [ 69 ]. Furthermore, it has been shown that the immune system can deteriorate with age, with a consequent decline in ovarian function [ 71 ]. These findings suggest a strong association between the immune system and the function of ovaries. Bukovsky put forward that the thymus was the largest immune organ in the body, and follicular depletion speed in mice with thymic removal was faster [ 70 ]. In ovaries, immune-related components, such as MDCs, T cell, and B cell, have significant effects on ovarian function maintenance. MDCs could stop cellular differentiation and keep cells in quiescence, in case of over-differentiation. However, due to immune system degeneration by age or diseases, ovaries occurred morphostasis and dysfunction, leading to premature ovarian failure or primary menopause [ 71 ]. Although the linkage between the functions of ovarian and the immune system was identified as early as 1970s, less focus has been placed on the relationship between the nests of FGSCs and the immune system. However, the fate of FGSCs mainly relies on immune cells and cytokines. There are many uncommitted MDCs and T cells in ovaries, which could recognize FGSCs. T cells differentiate into ovarian memory cells (OMC) and transmit ovarian information into lymph tissue. When niche degenerated, T cells would not produce OMC anymore. Therefore, symmetric and asymmetric division of FGSCs cannot be triggered [ 70 , 71 , 72 ].

Macrophages, play an importance role in maintaining the stable state of the stem cell niche. Macrophages produce numerous cytokines, both IL-10 and TNF-α, which speed up the clearance of aging erythrocytes and dead tissue. Therefore, macrophages may contribute to the senescence of the stem cell niche [ 73 ]. Furthermore, several cytokines, such as TNF-α and IL-1, can facilitate the processes of ovulation and angiogenesis. Animal model experiments have showed that TNF-α can facilitate vasculogenesis by increasing the expression of vascular endothelial growth factor in developing corpus luteum and inhibits angiogenesis in mature corpus luteum. Howere, this dual-regulatory mechanism of TNF-α remain to be elucidated [ 74 ]. Neverthless, the microenvironment of inflammatory can significantly impair the stem cell niche [ 75 ]. We found IL-2 and TNF-ɑ rose in senescent FGSCs while anti inflammatory factors TGF-βand IL-10 declined(unpulished). In the genetic level, the expression of MVH, Oct-4 and anti-aging gene SITR-1 and SIRT3 decreased, and aging gene P21 and P53 associated protein express increased. Those evidences indicated chronic inflammatory reaction in niche played an essential role in FGSCs senescence [ 55 , 76 , 77 , 78 ]. Thus, related immune cells and molecules from the ovarian stem cell nests are engaged in the asymmetric/symmetric division of OGSCs, migration, primordial follicle and neo-granulosa cell formation, and follicle maturation.

Hypoxia, oxidative stress injury and FGSCs aging

As mentioned above, the stem cell niche determines stem cell aging and remained stem cell numbers. Stem cell niche, combined with exogenous microenvironment alterations, such as changes from oxgen tension, temperature, hormones or cytokines from blood supplement, results in estricted self-renewal, senesence, skewed differentiation and compromised regeneration.The endogenous mechanisms inducing aging include telomere shortening, DNA damage accumulation, abnormal gene expression, epigenetic alteration, abnormal cell signaling [ 1 ] (Fig.  1 ). In the exogenous microenvironment, special focus has to be placed on the role of hypoxia in inducing and accelerating stem cell aging. Hypoxia, the unbalance between oxygen supply and demand, is the primary culprit of oxidative stress and chronic inflammation. Unfortunately, ovary is a deeply hypoxic organ due to it’s unique structure and cell compositon. On the one hand, with the growth and progression of follicular oocytes and the proliferation and division of granulosa cells, the oxygen demand gradually increases. On the other hand, continuous ovulation results in the increase of fibrous connective tissue and the significant reduction of blood vessels in the ovary, which leads to the decrease of oxygen supply in the ovary, and the decrease of blood vessels and blood supply in the ovary with the increase of age. In addition, chronic, low-grade inflammatory response caused by repeated ovulation and the accompanying oxidative stress aggravate the imbalance between supply and demand, resulting in low oxygen concentration in the ovary to about 1.3%-5.5% [ 79 , 80 ]. This may be the important reason that the speed of ovarian aging should be obviously faster than other organs by roughly 20–30 years earler. Molinari's transcriptomic analysis of human cumulus cells also revealed that hypoxia is a marker and an important determinant of follicular aging [ 81 ]. Hypoxia increases glycolysis metabolism,through hypoxia inducible factor-1 α (HIF-1 α), hypoxia activates monocytes/macrophages and TH1/TH17 cells through nuclear transcription factor NFκB (NFκB binding site exists in HIF-1 α promoter) and promotes the output of inflammatory factors like IL-6, IFN7 andTNF-α, resulting in chronic ovarian inflammation. Hypoxia also induced overproduction of ROS, which leading to damage to the ovaries from oxidative stress, oxidation of biomolecules including lipids, DNA and proteins,. Oxidative DNA damages, in turn, promote the formation of DNA adduct compounds such as 8-OXo-7, 8-dihydro-2 '-deoxyguanine and 4-hydroxynonenal. Both of them, together with inflammatory factors, cause the accumulation of DNA damage, abnormal gene expression, epigenetic alteration, dysregulation of cell signaling pathways and so on, leading to cell and organ aging. Many studies support a vicious cycle of oxidative stress-chronic inflammation: ROS increases due to oxidative stress, mediating through nod-like receptor 3 and NFκB to cause a range of inflammatory responses, for example, increased production of IL-1β, IL-6 and TNF-α, which in turn promote oxidative stress to accelerate aging [ 82 ]. Recently, Wang’ s team used high precision single-cell transcriptome sequencing technology for the first time to draw a compairing map of cynomolgus monkey ovarian cell aging and human ovarian cell It was found that aging leads to the imbalance of cell type-specific REDOX regulatory network in ovary and the decline of antioxidant capacity with aging, which is one of the main characteristics of ovarian aging in primates [ 3 ].

Hypoxia-induced chronic inflammation and oxidative stress on stem cell senescence. Hypoxia promotes the generation of inflammatory factors such as TNF-α, IFN7 and IL-6 through hypoxia-inducible factor-1 alpha (HIF-1 α), leading to chronic ovarian inflammation, and also induces excessive production of ROS, leading to the formation of DNA adducts such as 8-OXo-7, 8-dihydro-2'-deoxyguanine and 4-hydroxynonenal. Together with inflammatory factors, these two substances cause accumulation of damage to DNA, changes in epigenetics, abnormalities in gene expression, dysregulation of cellular signaling pathways, etc., ultimately leading to aging of cells and organs

As mentioned above, inflammatory factors can cause FGSCs aging, and oxidative stress also has an essential role in the aging of FGSCs. The concept of oxidative stress (OS) came up by Sohal in 1990, defining an adverse stress reaction that redundancy reactive oxide species produced by an imbalance of oxidative and anti-oxidative systems exceed eliminating ability. ROS is a chemically reactive oxygen atom or group of atoms produced during cellular metabolism. It could bi-directionally regulate cellular proliferation and apoptosis, where proper ROS concentration contributed to the activation of proliferative growth factors and their transduced signal molecules [ 83 ]. On the other hand, OS, in overdose situations, induce stem cell injury and multiorgan failure. For example, too much ROS caused skeletal muscle stem cell necrosis as pathological muscle injury and decreased hematopoietic stem cell activation [ 84 ]. Meantime, OS induces accumulation of malondialdehyde and ROS decreased expression of the anti-oxidative enzyme, injury mitochondria, inhibition of ovarian granulosa cell development. It also could cause ovarian inflammation reaction and reduce ovarian function, leading to infertility [ 85 ]. Excessive oxidative stress damage has been reported in the ovaries of chemotherapy-induced senescent mice, and elimination of oxidative stress damage promotes survival [ 86 , 87 ].

The strategy for delaying FGSCs aging

Rebuilding the nichs of fgscs.

One of the major challenges facing researchers and clinicians is successfully implementation of ovarian tissue engineering techniques to reconstruct the ovarian microenvironment and promote the initiation and maturation of primitive follicles. Tilly is optimistic about the future that rebuilding the habitable microenvironment of FGSCs for elderly women so that they could develop into mature eggs. He believe that this techeque will show great value in the future of assisted fertility technology [ 29 ]. Since alginate beads were first used in humans as artificial pancreas carriers in the 1980s, alginate polymers, because of it’s non-toxic, good elasticity, moderate solubility, transparency, long-term duration after implantis widely used in medical transplantation treatments. The channels within the hydrogel can promote the host cell invasion and form new vascular system, similar to the extracellular matrix (ECM).The gel pore network of ECM allows the combined nutrients and wastes to be delivered to tissues in a natural way, showing broad application prospects in 3D cell culture in vitro and the development of artificial organs in vivo [ 88 , 89 ]. Whether this technique can be used to reconstruct the microenvironment of FGSCs proliferation and differentiation is a topic of great significance and clinical application value. Recently, we selected alginate as the scaffold for FGSCs proliferation and differentiation, which can be connected with a variety of active substances such as melatonin, and promote the proliferation and differentiation of FGSCs by long-term and stable reconstruction of an ovarian microenvironment that is physiologically equivalent to that of healthy women of childbearing age, so as to rebuild the nichs of FGSCs and preserve female fertility (unpublished).

Anti-chronic inflammation and oxidative stress

Chronic inflammation and OS could trigger FGSCs aging, therefore, anti-chronic inflammation and OS drugs with fewer side effects and favorable biocompatibility are in consideration.

Resveratrol (RES)

RES, a natural component rich in grapes, mulberries, and other plants [ 90 ], can exert a variety of pharmacological functions, including anti-oxidation, anti-inflammatory, immune regulation, cell protection, anti-tumor, and anti-apoptosis effects [ 91 , 92 , 93 , 94 , 95 , 96 , 97 ]. RES effectively removes the accumulation of ROS. Rat follicular cells were shown to be free from cisplatin-induced oxidative toxicity after removal of excess ROS [ 98 ]. RES treatment can be used to improve the function of ovarian follicles by decreasing TNF-α level, confirmed by reducing the level of LH and the ratio of LH/FSH, which serve as indicators of ovarian function [ 99 ]. Jiang found that RES dramatically enhanced body weight and ovarian index, as well as follicle number and reduced follicular atresia in POF mice. Higher levels of Mvh, Oct4, SOD2, GPx and CAT were measured after in vivo and in vitro treatment with RES. RES therapy caused a significant decrease in the concentrations of TNF-α and IL-6 and an increase in IL-10 in the ovaries. In FGSCs, Mvh, Oct4 and SOD2 concentrations were higher and TNF-α, IL-6 and MDA concentrations were lower in the RES group. In summary, RES efficiently enhanced ovarian function and FGSCs in animal models of POF by reducing oxidative stress and inflammation, indicating that RES is a potential drug against POF by promoting the survival of FGSCs [ 87 ]. The therapeutic effect of RES on ovarian aging may depend on promoting the function of FGSCs and improving the ecological niche of FGSCs.

Chitosan oligosaccharide (COS)

 COS is an absorbable degradation of chitosan. It has been proved that COS had the functions of anti-oxidation, bacteriostasis, hypoglycemic and obesity contro l [ 100 , 101 ]. Our studies demonstrated that when the mice were given chitosan oligosaccharide by gavage for one month, the visceral coefficients of ovaries, the number of ovarian follicles, estrogen level and immune function enhanced. Meantime, the expression of the FGSCs marker increased, which was dependent on the COS dose, and also found chitooligosaccharides could directly promote the proliferation of FGSCs in vitro (unpublished). Our findings indicate COS has protective effects on ovaries through directly promote the proliferation of OGSCs or indirectly by enhancing the immune system.

Proanthocyanidin (PC)

PC is indispensable flavonoids in the human nutrition diet derived from grape seeds and pericarps. It has antibiotic, antioxidative and anticancer effects [ 102 ]. By now, there were few reports about the direct influence of PC on FGSCs, but the improvement of PC on ovarian aging has been studied [ 103 , 104 ].Therefore, it was speculated that the PC had a beneficial effect on FGSCs by its anti-oxidative stress and anti-aging, but further researches are needed.

The role of traditional chinese medicine

Traditional Chinese medicine

Most complications of ovarian aging are linked directly to ovarian hormonal deficiencies. Hormone replacement therapy (HRT) continues to perform a central role in the therapy of ovarian aging. However, HRT increases health risks for some patients, including breast cancer and cardiovascular disease [ 105 ]. As a result, numerous patients are switching to complementary and alternate medicine (CAM). As one major branch of CAM, traditional Chinese medicine (TCM), regarded as having less side effects, has been extensively used in China and in other countries to treat ovarian aging [ 106 , 107 , 108 ]. According TCM theory, ovarian aging diseases i are related to kidney, liver, spleen, heart, lung and other zang-organs’ mulfunction(we need to declare specifically that in the TCM theory, “kidney, liver, spleen, heart, lung”are not as same as the cencepts of morden medicine), espectially uterus mulfunction; dysregulation of thorough fare and conception vessels is the key link of ovarian aging pathogenesis.The etiology and pathogenesis concludes cold evil invasion, qi and blood deficiency, liver stagnation and qi stagnation, phlegm and blood stasis block, which eventually lead to meridian block, stagnation of qi and blood activities, zang-fu dysfunction. Therefore, the treatment of POF mainly starts from regulating throughfare and conception vessels, supplementing kidney and replenishing essence, smoothing liver and regulating qi, strengthening spleen and nourishing blood, promoting blood circulation and collaterals.

Acupuncture

Acupuncture is the treasure of Chinese culture and has been recognized, accepted, applied and popularized in the world [ 109 ]. At present, acupuncture has treated more than 800 kinds of diseases, and about 30% to 40% of them have significant curative effects [ 110 ]. In particular, acupuncture can significantly improve ovarian aging and ovarian function of POF and regulate menstrual cycle with clinical symptom improvement. Compared with hormone replacement therapy, acupuncture has good efficacy, lower recurrence rate, and less side effects, resultant earier accepted by patients and broader application prospects [ 1 , 111 , 112 , 113 ]. Meridians and collaterals spread all over the body, linking interio organs and body surface, maintaining the body's interior stability. Chong ren, nexus of the twelve meridinans on the surface, crosslink the uterus in the deep part of body, therefore affecting menstrual activity. Acupuncture stimulations on throughfare and conception vessels, Du Ren meridinans can tonify kidney and essence, regulat liver and qi, invigorate spleen and nourishing blood, activat blood and dredge collaterals, stimulat the functions of viscera, achieving the dual purpose of regulation.

Although Chinese traditional medicine has been widely used in the treatment of ovarian aging due to its precise curative effect, the mechanism of its treatment of ovarian aging is still far from clear. Whether it is related to the regulation of the function of FGSCs deserves further study.

Ovarian physiological or pathological failure results in the follicle reduction and terminating ovulation. The essence of the follicle reduction is the decreased number of FGSCs after follicles depletion, which is influenced by many factors like stem cell niche, inflammatory factors, hypoxia, oxidative stress injury. The immune cells and cytokine are key players of the stem cell niche. Immune dynfunction not only directly reduced ovarian resistance, due to FGSCs being more vulnerable to adverse external factors, but also indirectly hindered FGSCs proliferation by causing FGSCs niche defects. Therefore, exploring the mechanism of FGSCs aging is helpful in solving female infertility fundamentally in clinical practice. Heretofore, some studies have come up with solution that partially replenish exhausted FGSCs, via rebuilding the nichs of FGSCs, altering the microenvironment of chronic inflammation and oxidative stress. Whether the effect of Chinese traditional medicine on FGSCs in delaying ovarian aging remains to be further studied.

Availability of data and materials

Not applicable((review article).

Abbreviations

• Female germline stem cells

Premature ovarian failure

Very small embryonic-like stem cells

Follicle-stimulating hormone

Zona pellucida 2

E2 receptor-α

Stimulated by retinoicacid gene 8

Bone morphogenetic protein

Monocyte-derived cells

Ovarian memory cell

Hypoxia inducible factor-1 α

Oxidative stress

Extracellular matrix

Resveratrol

Chitosan oligosaccharide

Proanthocyanidin

Hormone replacement therapy

Complementary and alternate medicine

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This work was supported by the Natural Science Foundation of Jiangxi province (No.20181ACB20018); Basic Research Scheme of Shenzhen Science and Technology Innovation Commission(JCYJ20190812161405275);the National Nature Science Foundation of China (No. 81671455,31460307).

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Wenli Hong, Yasha Zhu, Jun’e Wu, Li Qiu, Shuyi Ling, Ziqiong Zhou, Yuqing Dai, Zhisheng Zhong & Yuehui Zheng

Shenzhen University Health Science Center, Shenzhen, Guangdong, 518000, People’s Republic of China

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Wenli Hong: literature review and manuscript writing, Baofeng Wang: manuscript writing, checking references and editing, Yasha Zhu: literature review and manuscript writing, Jun’e Wu: literature review and manuscript writing, Li Qiu: literature review and manuscript writing, Shuyi Ling: literature review and manuscript writing, Ziqiong Zhou: literature review and manuscript writing, Yuqing Dai: literature review and manuscript writing, Zhisheng Zhong: manuscript writing, checking references and editing, Yuehui Zheng: manuscript writing, checking references and editing. All authors read and approved the final manuscript.

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Hong, W., Wang, B., Zhu, Y. et al. Female germline stem cells: aging and anti-aging. J Ovarian Res 15 , 79 (2022). https://doi.org/10.1186/s13048-022-01011-2

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The Main Mechanisms of Mesenchymal Stem Cell-Based Treatments against COVID-19

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• Published: 04 April 2024

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• Jinling Li 1 , 3 ,
• Shipei He 1 , 2 ,
• Hang Yang 1 , 2 ,
• Lizeai Zhang 1 ,
• Jie Xiao 1 , 2 ,
• Chaoyi Liang 1 , 2 &
• Sijia Liu   ORCID: orcid.org/0000-0002-4050-3548 1 , 2  

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Coronavirus disease 2019 (COVID-19) has a clinical manifestation of hypoxic respiratory failure and acute respiratory distress syndrome. However, COVID-19 still lacks of effective clinical treatments so far. As a promising potential treatment against COVID-19, stem cell therapy raised recently and had attracted much attention. Here we review the mechanisms of mesenchymal stem cell-based treatments against COVID-19, and provide potential cues for the effective control of COVID-19 in the future.

Literature is obtained from databases PubMed and Web of Science. Key words were chosen for COVID- 19, acute respiratory syndrome coronavirus 2, mesenchymal stem cells, stem cell therapy, and therapeutic mechanism. Then we summarize and critically analyze the relevant articles retrieved.

Mesenchymal stem cell therapy is a potential effective treatment against COVID-19. Its therapeutic efficacy is mainly reflected in reducing severe pulmonary inflammation, reducing lung injury, improving pulmonary function, protecting and repairing lung tissue of the patients. Possible therapeutic mechanisms might include immunoregulation, anti-inflammatory effect, tissue regeneration, anti-apoptosis effect, antiviral, and antibacterial effect, MSC - EVs, and so on.

Mesenchymal stem cells can effectively treat COVID-19 through immunoregulation, anti-inflammatory, tissue regeneration, anti-apoptosis, anti-virus and antibacterial, MSC - EVs, and other ways. Systematically elucidating the mechanisms of mesenchymal stem cell-based treatments for COVID-19 will provide novel insights into the follow-up research and development of new therapeutic strategies in next step.

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Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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This study was funded by The National Nature Science Foundation of China (Grant No. 81860256), Fok Ying-Tong Education Foundation for Young Teachers in the Higher Education Institutions of China (Grant No. 171098) and Youth Science Foundation of Guangxi Medical University (Grant No. GXMUYSF 202319).

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Jinling Li, Shipei He, Hang Yang, Lizeai Zhang, Jie Xiao, Chaoyi Liang & Sijia Liu

Guangxi Colleges and Universities Key Laboratory of Biological Molecular Medicine Research, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Guangxi Medical University, Shuangyong Road, Nanning, 530021, Guangxi, People’s Republic of China

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Li, J., He, S., Yang, H. et al. The Main Mechanisms of Mesenchymal Stem Cell-Based Treatments against COVID-19. Tissue Eng Regen Med (2024). https://doi.org/10.1007/s13770-024-00633-5

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Immersed in a reservoir of potential: amniotic fluid-derived extracellular vesicles

• Ishara Atukorala   ORCID: orcid.org/0000-0003-0194-5877 1 , 2 ,
• Natalie Hannan 1 , 2 &
• Lisa Hui 1 , 2 , 3 , 4  

Journal of Translational Medicine volume  22 , Article number:  348 ( 2024 ) Cite this article

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This review aims to encapsulate the current knowledge in extracellular vesicles extracted from amniotic fluid and amniotic fluid derived stem/stromal cells. Amniotic fluid (AF) bathes the developing fetus, providing nutrients and protection from biological and mechanical dangers. In addition to containing a myriad of proteins, immunoglobulins and growth factors, AF is a rich source of extracellular vesicles (EVs). These vesicles originate from cells in the fetoplacental unit. They are biological messengers carrying an active cargo enveloped within the lipid bilayer. EVs in reproduction are known to play key roles in all stages of pregnancy, starting from fertilisation through to parturition. The intriguing biology of AF-derived EVs (AF-EVs) in pregnancy and their untapped potential as biomarkers is currently gaining attention. EV studies in numerous animal and human disease models have raised expectations of their utility as therapeutics. Amniotic fluid stem cell and mesenchymal stromal cell-derived EVs (AFSC-EVs) provide an established supply of laboratory-made EVs. This cell-free mode of therapy is popular as an alternative to stem cell therapy, revealing similar, if not better therapeutic outcomes. Research has demonstrated the successful application of AF-EVs and AFSC-EVs in therapy, harnessing their anti-inflammatory, angiogenic and regenerative properties. This review provides an overview of such studies and discusses concerns in this emerging field of research.

Introduction and background

Composition of amniotic fluid.

Amniotic fluid (AF) is a unique conditioning medium for the developing fetus throughout gestation until birth [ 1 ]. The composition and volume of AF changes across gestation and aligns with key gestational stages [ 2 ]. The AF volume increases linearly from first trimester until about 33 weeks gestation and then reduces towards full-term [ 3 ]. It starts as a by-product of maternal serum consisting of water and electrolytes and gradually changes to fetal products by the late second trimester [ 1 , 4 , 5 , 6 ]. In the early weeks of gestation, the fetal skin is a simple epithelium layer, as such AF freely diffuses across [ 5 ]. However, after keratinization completes, around week 25, fetal urination becomes the main source of increasing AF volume, while fetal lung secretions also contribute significantly [ 3 ]. Fetal “respiration” and swallowing remain the principal routes for AF resorption [ 3 , 7 ]. At term, the human fetus produces 800–1200 ml of urine per day, which can replace the entire AF volume within 12–24 h [ 8 , 9 ].

AF is rich in numerous nutrients and growth factors supporting fetal development [ 10 ], while antibodies and antibacterial agents present within the fluid help to protect the fetus from infections [ 11 ]. Apart from playing an integral part in fetal health, AF has been a useful prenatal diagnostic sample, since amniocentesis was first performed in the late 1960s for fetal karyotyping [ 1 ].

What are extracellular vesicles?

Extracellular vesicles (EVs) are lipid-bilayer membrane-enclosed vesicles that are secreted by virtually all cells [ 12 ]. Their diameter can range from small EVs of 30–150 nm to oncosomes of 10 µm [ 13 ]. Since the first description of EVs in the 1980s [ 14 , 15 ], EVs have been extensively researched in health and disease. There are many classes of EVs, including exosomes, oncosomes, shedding microvesicles, migrasomes and apoptotic bodies. The categorisation is based on their biogenesis and secretion mechanisms, size, and function [ 16 , 17 , 18 ]. EVs secreted by the host cells can mediate both proximal and distal signalling events in organisms [ 19 , 20 , 21 ]. Their biological cargo is transported intact, avoiding degradation through the protection of the lipid bilayer membrane [ 22 ]. Their unrestrictive crossing of the blood–brain barrier makes them an appealing delivery mode for central nervous system therapeutics [ 23 , 24 ].

EVs as a method of studying human reproduction

EVs have been a valuable source of information about human reproduction. Examples include uterine luminal fluid EVs in fertilisation, maintaining the sperm viability in the oviduct and continuity of pregnancy by keeping Ca 2+ homeostasis [ 25 ]. The potential influence can be attributed to their selectively packaged cargo [ 26 ]. They appear to play a critical role in embryo implantation, establishing the first communication between the mother and the conceptus [ 27 , 28 ]. Placental EVs are known to influence uterine spiral arterial remodelling under physiological conditions, but might be compromised under pathological conditions [ 29 ].The role of AF-EVs in parturition [ 30 , 31 ] is discussed later in detail.

It is evident that the molecular signature of AF-EV cargo changes according to feto-maternal pathologies, creating opportunities for many clinical applications. Pregnancy complications such as pre-eclampsia [ 32 ] and preterm labour [ 30 , 33 ], fetal complications such as congenital hydronephrosis [ 34 ] and fetal alcohol syndrome [ 35 ] have been studied using AF-EV borne molecules, which are discussed later in detail. While these studies are beneficial in biomarker discovery and knowledge gain, they are yet to achieve clinical translation.

Amniotic fluid EVs and amniotic fluid stem/stromal cell EVs in therapy

Therapeutic applications of EVs have been investigated by researchers, mostly as drug delivery vehicles [ 23 , 24 , 36 ]. However, AF-EVs and AFSC-EVs are more than a transport mode for exogenous therapeutics. They are loaded with endogenous molecules with therapeutic potential, that can influence tissue regeneration, anti-inflammation, paracrine signalling, and immunomodulation [ 37 , 38 ]. Unmodified EVs isolated from term AF have been tested in pre-clinical models to treat conditions such as bronchopulmonary dysplasia [ 39 ] and azoospermia [ 40 ]. They have also been used in human trials to treat severely ill COVID-19 patients. Case studies performed in the USA demonstrated the safe clinical use of AF-EVs in humans, successfully improving lung function of intubated COVID-19 patients [ 41 , 42 ].

EVs derived from amniotic fluid stem cells/stromal cells (AFSC-EVs) are a popular choice for therapeutic experimentation in pre-clinical models, owing to the easy access to the source material and successful laboratory production. The studies included in this review used several distinct terms to identify the cell populations—stem cells, mesenchymal stem cells and mesenchymal stromal cells . The field of stem cell research acknowledges the potential ambiguity in cell nomenclature by various research groups [ 43 , 44 , 45 ]. Therefore, for the purpose of this review, we have used AFSC-EVs to identify EVs derived from the conditioned media of all three different cell types mentioned.

EVs from AF stem cell cultures appear to have a more consistent paracrine profile than stem cells, thus avoiding the unpredictability that is tied with stem cell therapy [ 38 ]. AFSC-EVs have produced positive responses in preclinical studies of various pathologies, including premature ovarian failure [ 46 ], cardiac injury [ 47 , 48 ], neuroinflammation [ 49 , 50 ] and necrotising enterocolitis [ 51 , 52 ].

The aim of this narrative review is to summarise the current knowledge of AF-EVs and AFSC-EVs, including their isolation and characterisation, physiological and pathological implications, and potential clinical applications. Due to the variability in methods used to isolate EVs, studies discussed in this review include a wide range of EV sizes and categories with varying molecular properties, including microparticles, microvesicles, exosomes and nanovesicles (Table  1 ).

Selection of studies

PubMed Central was searched on the 13th of June 2023, using the keyword combination (exosomes OR extracellular vesicles) AND amniotic fluid, using the advanced search option. A total of 148 search results published from 2000 to June 2023 was retrieved. Articles were included if they were full manuscripts published in English reporting original research on EVs directly isolated from AF or from AF stem cell cultures.

A list of 74 articles was selected for full-text review after screening of titles, abstracts, and keywords, of which 7 irrelevant studies were excluded. Two articles were retrieved after a manual search of reference lists of included articles. A total of 69 full-text articles were included (Additional file 1 . List of included studies) (Fig.  1 ). Forty-four (64%) studies were published since 2020. We performed a narrative overview and content synthesis of the final included articles.

PRISMA flow chart of the study selection criteria for the review. A thorough literature search via NCBI Pubmed resulted in 148 articles, of which 69 were included in this review, after excluding irrelevant studies

AF-EV isolation

The source of af.

The majority of studies derived human AF samples from clinically-indicated amniocentesis (18), term labour or Caesarean section (13). Three studies did not state the source of AF. Two other groups studied murine and ovine AF (Table  2 ).

Lack of standardization in AF-EVs isolation methods

The most common method to isolate small AF-EVs was differential centrifugation coupled with ultracentrifugation. The majority of studies performed centrifugation at 300 g for 15 min to remove cells, followed by 2000 g for 20 min to eliminate cellular debris. This step was most commonly followed by centrifugation at 10,000 g for 30 min and filtration to remove larger vesicles. Ultracentrifugation at 100,000–120,000 g for varying time periods pelleted down small EVs.

Various methods were reported for further purification of EVs following ultracentrifugation. While some researchers opted for density gradient centrifugation or ion exchange chromatography, others used commercially available kits for EV isolation (Table  2 ). Researchers preferred amniocentesis for sample collection over Caesarean section and differential centrifugation for EV isolation as indicated in Table  3 (a summary of Table  2 ).

Ebert and Rai developed an unconventional three-step centrifugation protocol to isolate AF-EVs, that involved addition of dithiothreitol (DTT) to the EV pellet to denature external protein aggregates [ 53 ]. This method may not be suitable for studies focusing on EV membrane proteins as DTT can denature the ectodomains of proteins. Others used a centrifugation-based method in combination with filtration and commercially available chromatography columns for EVs isolation from small volumes (down to 250 µL) of AF [ 54 ]. A comparison of methods study stated that ultracentrifugation resulted in better EV yield from human AF than commercial exosome isolation reagents [ 55 ].

The variability in methods may partly be due to the variability in samples. For example, term AF contains vernix caseosa (white wax-like substance covering the fetal skin) compared to second trimester AF, requiring strenuous sample cleaning steps. While AF can be a challenging sample, one would expect to have largely consistent methods for EV isolation from conditioned media derived from cell cultures.

Amniotic fluid stem/stromal cell EV isolation

Amniotic fluid stem/stromal cell cultures are used as a reliable supply of evs.

Many researchers have isolated AF stem or stromal cells and cultured them to provide a convenient and continuous in vitro source of EVs. These studies used human/murine primary or cryopreserved cells obtained from second-trimester amniocentesis, elective Caesarean sections or both. Five research groups obtained mouse AF stem cells (Table  4 ), presumably to maintain the consistency with experimental animal models. Table 5 summarises this information, providing a count of studies that used different sample sources and EV isolation methods.

Stem cells were most commonly isolated from AF by fluorescence activated cell sorting for c-Kit expression [ 47 , 48 , 52 , 56 , 57 , 58 ] or for CD44/CD105 expression [ 59 ]. Other researchers cultured cells from AF and separated the colonies based on the fibroblast morphology of the cells [ 60 , 61 ]. Whether these different methods impact EV biogenesis and secretion pathways differently in stem cells is yet to be understood.

Majority (79%) of the AFSC-EV studies included in this review referred to their cell populations as stem cells while 2 studies mentioned the isolation of mesenchymal stromal cells. Five other studies mentioned the use of mesenchymal stem cells. Table 4 describes different culture conditions used by research groups to grow the isolated cells.

A variety of isolation methods for AF stem/stromal cell EVs

There is a variety of methods of EV isolation from AF stem cell-conditioned media, but most employed some form of differential centrifugation with many variations in the centrifugation steps. Studies published in the past 2–3 years commonly used the classic approach of differential centrifugation steps to remove live and dead cells (500 g ), cell debris (2000 g ), large vesicles (10,000–15,000 g ) and a final ultracentrifugation collecting small EVs (100,000–120,000 g ) (Table  4 ). A recent study comparing ultracentrifugation and a novel polyethylene glycol (PEG)-based EV precipitation method demonstrated that PEG-based isolation produced approximately five times more EV yield and EV proteins, but one third the EV-RNA content compared to ultracentrifugation [ 62 ]. The choice of isolation method may consequently influence the properties of EVs [ 62 ].

Isolation methods depend on the differential density, solubility factors and size of the target EVs [ 63 ]. Efforts to standardize EV research by the International Society for Extracellular Vesicles is reflected in the studies published since 2020, with a degree of consistency in methods compared to earlier studies. However, all methods result in some degree of variation in size range, purity and protein content of each EV preparation. Some research groups have attempted to standardize their laboratory protocols by adhering to good manufacturing practices (GMP) guidelines [ 41 , 42 , 64 ], or used GMP-grade AF stem cells for culture [ 65 ]. This is an essential step in ensuring that the findings from basic research can eventually be translated into clinical applications and scaled up into commercial products.

Characterisation of EVs should adhere to internationally accepted guidelines

The established guideline for characterising EVs and confirming their successful isolation is the Minimal Information for Studies of Extracellular Vesicles (MISEV2018) statement approved by the International Society for Extracellular Vesicles [ 66 ]. This characterization involves three main steps: (i) nanoparticle tracking analysis to confirm the size range and concentration of the isolated vesicles, (ii) transmission electron microscopy to visualise their morphology, and (iii) screening for standard EV enriched markers such as Alix, TSG-101 and tetraspanins CD63, CD81 and CD9 (Fig.  2 ). Only 23 (36%) of the included studies employed all three characterisation methods.

Commonly employed EV isolation and characterisation methods. Human/animal AF or conditioned media of AF stem cell/MSC cultures are first subjected to differential centrifugation to remove cellular debris. The supernatant is subjected to ultracentrifugation/size-exclusion chromatography/affinity chromatography or a combination of these methods. An optional further purification of the isolated EV population is achieved using density gradient centrifugation, filtration, or ion-exchange chromatography. Isolated EVs are characterised using nanoparticle tracking analysis for EV concentration and size range, transmission electron microscopy for EV morphology and Western blotting to analyse EV protein markers. Figure created with BioRender.com

Amniotic fluid EVs are abundant and immunologically active

Human AF appears to be a more concentrated source of EVs compared to other bio-fluids, with AF-EVs concentrations up to 41-times higher than maternal plasma [ 67 ]. AF-derived exosomes are also reportedly smaller (~ 100 nm) than EVs of other sources and contain standard EV markers [ 54 ]. The predominant fetal renal origin of these vesicles has been suggested by the presence of tetraspanin CD24, kidney marker aquaporin-2 [ 68 ] and CD133 [ 32 ]. Other identified proteins in AF-EVs include an obscure, lower molecular weight CA125 species [ 69 ], tubulin and heat shock proteins Hsp72 and Hsc73 [ 70 ]. These extracellularly released heat shock-related proteins are known as alarmins and are expressed under hypoxic, immune or inflammatory stress conditions [ 71 ].

AF-EVs are known for their immunomodulatory properties, which can suppress T-cell activation and pro-inflammatory cytokine release in-vitro [ 72 ]. AF-EVs may act as both pro- and anti-inflammasome activating agents, potentially priming the fetal immunity owing to the presence of bacterial DNA in these vesicles [ 73 ]. Moreover, AF-EVs triggered epithelial-to-mesenchymal transition and myofibroblast activation in stem cells [ 74 ]. These studies have revealed important biological properties of AF-EVs, suggesting their many roles and potential uses.

AF stem/stromal cell-derived EVs are bioactive and have distinct ‘omic profiles

The AFSC-EV therapeutics is a rapidly growing field of research. One of the first studies exploring AFSC-EVs reported on their active immunoregulatory properties [ 75 ]. A recent comparative study confirmed a 25% higher EV yield from AF stem cells compared to human bone marrow-derived stem cells, making them preferable for clinical applications [ 76 ]. They contain a significant amount of the biologically active molecules of the secretome of AF stem cells. AFSC-EVs contain miRNA, but not mRNA, suggesting their role in directly or indirectly regulating existing signalling pathways of recipient cells rather than enforcing new ones [ 47 ].

Researchers have suggested that AFSC-EVs are metabolically independent entities [ 77 ]. Equivalently, EVs isolated from semen of multiple species (human, canine, equine, and bovine origin) produced ATP intrinsically through the glycolytic pathway [ 78 , 79 ]. Presence of active metabolic enzymes, particularly glyoxalases and MG-H1, in AFSC-EVs cargo [ 61 ] adds up to this concept.

AF-EVs contain anti-inflammatory, immunomodulatory, and free radical scavenging properties [ 39 ]. These functions are manifested by stabilizing telomere lengths [ 80 ], increasing cell adhesion and migration, and regulating cytokine production under inflammatory conditions [ 81 ] in recipient cells. These findings indicate that AF-EVs may indirectly modulate the maternal immune system, potentially preventing fetal rejection by the mother’s body.

Selecting the appropriate source of AF stem cells based on desired therapeutic outcome is essential as neonatal and perinatal AFSC-EVs possess distinct proteomic and transcriptomic profiles [ 82 ]. Second trimester amniocentesis-derived immature AFSC-EVs displayed pro-vasculogenic, pro-regenerative, and anti-aging properties, while term pregnancy-derived AFSC-EVs exhibited pronounced immune-modulatory and anti-inflammatory characteristics. However, both types of AFSC-EVs had a rich microRNA signature containing regenerative paracrine factors [ 82 ].

Amniotic fluid derived EVs as potential biomarkers

Exosomal shuttle rna and fetal development.

The RNA cargo in exosomes is known as exosomal shuttle RNA (esRNA) [ 83 ]. esRNA within AF-EVs is protected by the lipid membrane from digestion by nucleases, making transcripts readily available for diagnostic or prognostic purposes [ 22 ]. A number of biomarker discovery studies basing AF-EV esRNA have been published for fetal conditions such as congenital hydronephrosis [ 34 ], congenital diaphragmatic hernia [ 84 ], fetal alcohol exposure, osteogenic differentiation [ 35 ], congenital heart defects [ 85 ] and ureteropelvic junction obstruction [ 86 ]. However, these studies are yet to be translated into clinically useful predictors of perinatal outcomes.

AF-EVs and parturition

Labour is an inflammation driven process. Resident and infiltrating immune cells in reproductive tissue [ 87 , 88 ] and free cytokines in AF are associated with labour, both term and preterm [ 89 , 90 , 91 ]. Preterm labour, intra-amniotic inflammation and infection, all result in differential packaging of cytokines in AF-EVs [ 33 ]. Placental alkaline phosphatase (PLAP)/CD63 ratio in AF-EVs has been suggested as a marker for preterm birth and preterm premature rupture of membranes [ 30 ]. Others have postulated that fetal lung-derived EVs in AF may have a role in parturition, as they induced senescence-associated secretory phenotype and proinflammatory molecules in human amniotic epithelial cells in term pregnancies [ 31 ]. Moreover, transcription regulator HIF1α contained in AF-EVs impacts comparatively shorter interval between amniocentesis and parturition [ 92 ].

AF-EVs in obstetric complications

AF-EVs have been studied in a limited number of obstetric complications. Elevated CD105 (endoglin) in AF-EVs resembled augmented angiogenesis in preeclampsia [ 32 ]. Others studied AF-derived microparticles in disseminated intravascular coagulation and hypotension in amniotic fluid embolism [ 67 ]. These fetal-origin EVs [ 93 ] were predominantly from apoptotic events of epithelial and leukocytic cells [ 94 ]. Their cargo included procoagulant molecules such as phosphatidylserine and tissue factor [ 95 ], and extrinsic tenase complexes [ 96 ].

Congenital cytomegalovirus infection is a common infection worldwide and may result in a range of undesirable outcomes including fetal death [ 97 ]. Identification of the association between the fetal infection and the EV-borne pro-inflammatory cytokine profile [ 98 ], may be a step towards predictive biomarkers for severity of fetal infection.

While these studies have revealed potential AF-EV-borne biomarkers for obstetric complications, they are primarily discovery-phase reports that require to be clinically validated.

Therapeutic applications of AF-EVs and AFSC-EVs

AF and AF cell-derived EVs gained substantial interest as a therapeutic in regenerative medicine. Biological activity of these EVs is dependent on the treatment dose, rather than the specific size or purity of the isolated EV populations [ 99 ]. As a cell-free product loaded with bioactive molecules, they contain many desirable properties. EVs have been shown to modulate inflammation [ 58 , 100 , 101 , 102 ], curb oxidative stress [ 103 ] and augment wound healing [ 104 , 105 ], ultimately leading to tissue regeneration. Moreover, as a natural cell-derived product, EVs present advantages such as biocompatibility and minimal toxicity for recipients. A summary of the preclinical and clinical therapeutic studies retrieved from our literature search is presented in Table  6 .

AF is an accessible human fetal sample with significant biological value. However, until recently, it has been under-explored in reproductive medicine compared to other sources such as maternal plasma and placental tissue. Keller and colleagues first reported the detection of EVs in human and murine AF in 2007 [ 68 ], but the field remained quiescent until the past 4 years. There is an increased interest in AF derived biologics since 2020, making up for 64% of studies in this review.

Researchers have debated the optimal methods for EV isolation and their purity assessment for the last decade [ 63 ]. The community achieved consensus with the publication of the Minimal Information for Studies of Extracellular Vesicles guidelines [ 66 ] regarding basic isolation and characterization of EVs. However, EVs are a heterogenous group and cannot be separated by biogenesis using existing methods [ 18 ]. Therefore, nomenclature of the vesicles is challenging and will remain a discussion for the foreseeable future. At present, large EVs or small EVs seem to be the appropriate terms to describe an EV population, based on the employed isolation methods. Our review shows the inconsistent terminology (Table  1 ) used in reproductive EV research.

Researchers seem to prefer ultracentrifugation over other methods for AF-EVs and AFSC-EVs isolation (Tables 3 and 5 ). However, specific details such as durations of spins and speed were lacking in several studies. Ultracentrifugation is considered the “gold standard” method for EV isolation due to its reliability and optimal yield [ 106 , 107 ]. However, EV samples isolated using ultracentrifugation require further purification methods to achieve homogeneity. The use of other methods such as commercially available chromatography columns and polymeric precipitation were observed when sample sizes were too small for centrifugation. Many factors such as the source material and its volume, EV size range of interest and the downstream use of the isolated EVs can influence the isolation methods. Nonetheless, the choice of isolation method largely appeared to be at the discretion of individual research groups. A clear and globally accepted, robust set of guidelines for the methodologies for AF derived EVs would benefit this emerging research field.

The laborious nature of the differential centrifugation and ultracentrifugation procedures limits the scalability for EV production for clinical use [ 108 ]. Commercial products are attractive solutions but have not gained widespread acceptance as only 17% of studies in this review have utilized them. Methodological studies have compared the commercial EV isolation kits versus ultracentrifugation [ 55 ], and the use of both methods together in the same protocol [ 54 ] resulting in varying inferences. Regardless of these time and labour effective new commercial products, ultracentrifugation remains the preferred method for most researchers. Studies have presented EV concentrations using a range of units such as particles per gram of EV proteins, vesicles per millilitre of fluid (it is unclear if the fluid refers to AF or the EV suspension buffer) and EV proteins (µg) per millilitre. Adoption of a standard unit such as vesicle number per millilitre/gram of starting material (body fluid/tissue) or per million cells would help advance the field by allowing more direct comparisons of results and facilitating replication of studies.

EV isolation from conditioned media requires specific conditions. Use of serum-free culture media or EV-depleted FBS in the media is widely accepted, to avoid introducing exogenous EVs. Other components such as antibiotics, growth factors and supplements can also affect EV biogenesis and their cargo [ 66 ]. Confluence of cells, culture temperature, percentage CO 2 , O 2 and incubation time before EV isolation may all alter EV yield, quality and their biomolecule content [ 109 , 110 ].

Therefore, it is important all information is reported accurately in publications and lack thereof may result in lack of reproducibility. Many groups studied RNA cargo in EVs to develop predictive disease biomarkers. However, the effect of different EV and evRNA purification methods for downstream sequencing and profiling is not known [ 18 ]. Standardization of methodologies and terminology for publications is of central importance going forward. The compliance of experimental protocols with good manufacturing practice guidelines is highly commendable, which improves the quality of research and reproducibility across laboratories, facilitating smooth clinical translation.

Only one clinical application for AF-EVs has progressed to human clinical trials, no doubt accelerated by the urgency to develop novel therapies during the COVID-19 pandemic. Zofin, a human AF derivative enriched for EVs, is being evaluated in COVID-19 patients with severe acute respiratory syndrome in three separate studies, by the same group (NCT05228899, NCT04657406, NCT04384445). These clinical trials are still in progress, but pilot studies have proved safe use of AF-EVs with improved clinical outcomes.

The appeal of AF-EVs for COVID-19 treatment lies in their anti-inflammatory properties and their potential to curb the ‘cytokine storm’ of severe disease. Another clinical trial in Israel (NCT04747574) administered CD24-loaded EVs derived from HEK293 cells to COVID-19 patients, with encouraging outcomes [ 111 ]. Several other groups have also manifested the safety and feasibility of using acellular AF (not enriched for EVs) to treat COVID-19 patients in the clinic [ 112 , 113 ]. Treatments for other inflammatory diseases also have shown the capacity of both AF-EVs and AFSC-EVs to reduce inflammation, restoring tissues or cells to their homeostatic state.

The number of clinical trials using AF-EVs or AFSC-EVs is currently minimal. However, clinical trials have used processed or unprocessed AF to treat chronic wounds (NCT04438174), osteoarthritis (NCT03074526, NCT02768155, NCT04886960), stenosing tenosynovitis (NCT03583151) and venous stasis ulcer (NCT04647240) among many others. The need for expertise, purpose-built instrument and laborious nature of isolating EVs may have delayed AF derived EV research reaching clinical translation.

Regenerative properties of AF-EVs and AFSC-EVs were used to treat necrotizing enterocolitis, premature ovarian failure and wound healing [ 99 , 114 ]. Most studies demonstrated the desirable outcomes of these EV treatments in in-vitro and in-vivo models and some studies deciphered the underlying molecular mechanisms. In-depth understanding of the mechanisms will be beneficial in translating the findings to clinical applications. For example, AFSC-EVs treatment of cystinosis may have revealed a prospective targeted therapy for this rare disease, as the EVs were naturally loaded with cystinosin and reprogrammed the recipient mutant cells [ 115 ].

Stem cell-EV therapy has emerged as an attractive alternative to stem cell therapy, as it omits the challenges of unpredictable host rejection and poor efficacy. The shift in interest was promoted by research studies increasingly implying that the therapeutic effect of stem cells is mediated by the extracellular paracrine factors exerted via EVs [ 38 ]. Many research studies have demonstrated the successful utility of AFSC-EVs in pre-clinical models to treat different pathologies including necrotizing enterocolitis [ 51 , 52 , 100 , 101 ], hypoplastic neonatal lungs [ 65 , 116 , 117 ] and wound healing [ 104 , 105 ]. AF composition is dynamic and often represents the gestation-dependent development of fetal organs [ 118 , 119 ]. Accordingly, the careful choice of gestation for AF collection according to the intended purpose of EVs was observed in these studies (Fig.  3 ). For example, for lung function-related therapies, AF obtained from elective Caesarean sections at term was used for EV or stem cell isolation, as fetal lungs rapidly develop close to parturition [ 120 ]. For other conditions, such as treating wound healing and necrotising enterocolitis, researchers used samples from second-trimester amniocentesis, where the AF is rich with factors implicated in tissue regeneration.

Gestation of amniotic fluid is matched with intended therapeutic use. The gestation at which the AF was collected was often matched to the therapeutic purpose of the research studies/clinical trials. For example, second trimester AF derived EVs were used when the regenerative properties of EVs were desired whereas third trimester AF derived EVs were preferred for lung function therapies. Researchers obtained second trimester AF from amniocentesis and third trimester AF from labour/Caesarean section at term. Figure created with BioRender.com

Our understanding of the biological difference between AF-EVs and AFSC-EVs is narrow and therefore there is currently no definitive evidence to propose biological superiority of one over the other. They conceivably are not bioequivalent and cannot be used inter-changeably. This is a grey area that has not been looked at yet. Researchers seem to be interested in EVs from both sources alike. Thirty-four (49%) articles included in this review used AF-EVs while 35 (51%) used AFSC-EVs. Since AFSC-EVs originate from one cell type, presumably they have minimal batch variations and more predictable biological properties compared to AF-EVs—both beneficial properties for clinical use. Therefore, a comprehensive comparison between AF-EVs and AFSC-EVs can benefit their applications.

If these EVs clear the hurdles to become therapeutics, AF collection and processing mechanisms will need to be increased and standardised. Additional research is needed to assess the inherent variation in AF samples from different donors and the suitability of singular or pooled samples for clinical applications. Despite the great excitement, there is a real risk that many studies of EVs as prognostic markers or therapies may be lost in the ‘valley of death’ between preclinical studies and clinical trials [ 121 ]. Therefore, further research, together with standardisation, may immensely progress the translation of these findings into clinical applications.

Availability of data and materials

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.

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Atukorala, I., Hannan, N. & Hui, L. Immersed in a reservoir of potential: amniotic fluid-derived extracellular vesicles. J Transl Med 22 , 348 (2024). https://doi.org/10.1186/s12967-024-05154-2

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Advancements in culture technology of adipose-derived stromal/stem cells: implications for diabetes and its complications.

• 1 Department of Endocrinology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
• 2 Hubei Provincial Clinical Research Center for Diabetes and Metabolic Disorders, Wuhan, China

Stem cell-based therapies exhibit considerable promise in the treatment of diabetes and its complications. Extensive research has been dedicated to elucidate the characteristics and potential applications of adipose-derived stromal/stem cells (ASCs). Three-dimensional (3D) culture, characterized by rapid advancements, holds promise for efficacious treatment of diabetes and its complications. Notably, 3D cultured ASCs manifest enhanced cellular properties and functions compared to traditional monolayer-culture. In this review, the factors influencing the biological functions of ASCs during culture are summarized. Additionally, the effects of 3D cultured techniques on cellular properties compared to two-dimensional culture is described. Furthermore, the therapeutic potential of 3D cultured ASCs in diabetes and its complications are discussed to provide insights for future research.

1 Introduction

Stem cell-based therapy, including pluripotent stem cells (PSCs) and mesenchymal stromal/stem cells (MSCs), represents an innovative therapeutic strategy that capitalizes on the distinctive characteristics of stem cells, such as self-renewal and differentiation capabilities, to facilitate the regeneration of impaired cells and tissues within the body or the substitution of these cells with new, healthy, and fully functional cells by delivering exogenous cells ( 1 ).

PSCs are characterized as a type of self-renewing cells capable of differentiating into diverse cellular phenotypes originating from the three germ layers of the body ( 2 ). PSCs, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), has revolutionized stem cell research and cell-based therapy ( 3 ). Nonetheless, the utilization of ESCs is constrained by ethical considerations, the possibility of immunological rejection, and the potential for tumorigenicity ( 1 , 4 ). In contrast, iPSC technology overcomes ethical dilemmas associated with ESCs derived from human embryos, enabling the creation of patient-specific pluripotent stem cells. However, iPSCs are generated through the ectopic expression of pluripotency factors, often facilitated by viral vectors or non-viral reprogramming factors, which may lead to genomic instability ( 5 , 6 ). Besides, iPSCs have been shown to elicit T cell-dependent immune response ( 7 ) and promote tumor formation ( 3 , 8 ). Consequently, thorough safety assessments are imperative prior to iPSC transplantation.

Mesenchymal stromal/stem cells (MSCs) are adult stem cells with multipotent capabilities, including self-renewal (albeit limited in vitro) and differentiation into various mesenchymal lineages ( 9 , 10 ). MSCs have been shown to overcome ethical concerns and mitigate the risk of mutational side effects associated with. Additionally, MSCs exhibit the lowest immunogenicity compared to other stem cell types, making them a favorable option for clinical use ( 11 ). In the field of organ and cell transplantation, MSCs have been utilized for their secretion of growth factors and immunoprotective cytokines. Their ability to differentiate into various cell types has been harnessed for applications in tissue engineering ( 12 ). Among these, adipose-derived MSCs (ASCs) are particularly advantageous due to their larger storage with less discomfort and damage to the donor site, easier accessibility without significant donor site morbidity, higher proliferation ability, fewer ethical concerns, and fewer immunological rejection ( 11 , 13 , 14 ). Furthermore, some growth factors and immunomodulators are more actively secreted in ASCs ( 13 ). Therefore, ASCs may be a better candidate for clinical application in theory.

Diabetes mellitus (DM) is a severe and chronic disease characterized by elevated blood glucose levels resulting from aberrant islet β-cell biology and insulin action ( 15 ). In 2021, the global population living with diabetes reached 529 million ( 15 ). Given β-cell dysfunction across various types of DM, most patients ultimately require insulin therapy ( 16 – 18 ). However, this therapy is frequently limited by individual factors, such as weight gain, fear of needles and lifestyle considerations, all of which contribute to poor glycemic control. Furthermore, insulin therapy cannot reverse β-cell damage and progress of diabetes, or replicate the normal physiological state. In recent clinical applications, pancreatic islet and cell transplantation have emerged as potential strategies ( 19 ). However, these procedures have numerous challenges, including the scarcity of suitable donors, surgical complexities, side effects associated with immunosuppressive agents as well as exhaustion of transplanted organs and cells ( 11 ). Furthermore, it is necessary to maintain β-cell function and blood glucose homeostasis, otherwise life-threatening complications are likely to occur ( 20 ).

In the treatment of diabetes and its complications, ASCs have been used due to their inherent attributes such as self-renewal capacity, differentiation potential, homing mechanism and immunosuppressive property ( 11 , 21 ). Furthermore, three-dimensional (3D) cultured cells are studied to prolong the lifespan of transplanted cells and enhance their pro-healing functions in unfavorable environments ( 22 – 25 ). Recent literature provides numerous strategies for obtaining 3D cultured ASCs ( 26 ). These cells possess enhanced abilities to maintain their stemness and display multilineage plasticity compared to cells cultured in adhesion ( 26 ). Moreover, 3D cells more closely mirror biological processes compared to cells cultured in traditional monolayers, driving the need for the development of 3D culture, including spheroids, organoids, organ-on-a-chip models, and bioprinting ( 27 – 29 ).

Despite being an emerging and rapidly developing technology, there is currently no standardized method for ASC culture and no summary for the research of 3D cultured ASCs in diabetes and its complications. In this review, we summarize current knowledge about monolayer ASC culture techniques, with a particular emphasis on the influential factors during culture. Additionally, the effects for cellular properties of 3D cultured methods compared to two-dimensional (2D) culture is described. Furthermore, the therapeutic potential of 3D cultured ASCs in diabetes and its complications are discussed to provide insights for future research.

2 Nomenclature of adipose-derived stromal/stem cells

There is inconsistency in the nomenclature of this plastic adherent cell population isolated from adipose tissue ( 30 ). In 2006, the International Society for Cellular Therapy (ISCT) acknowledged the “inconsistencies and ambiguities” of the term “mesenchymal stem cells” and recommended a new designation: multipotent mesenchymal stromal cells ( 31 ). It is recommended to use the abbreviation “MSCs” in conjunction with extra information like AD-MSCs ( 9 ) (adipose tissue-derived MSCs) or MSC(AT) ( 32 ) and clearly define stem cells or stromal cells in terms of their function ( 9 ). Additionally, Caplan proposed the term “medicinal signaling cells” due to their therapeutic actions, which include homing to the site of injury and secreting regenerative and immunomodulatory factors ( 33 ). Despite the advocacy for standardization in nomenclature, it is still most common to refer to MSCs as “mesenchymal stem cells”, followed by “mesenchymal stromal cells” or a combined use of “stem/stromal” terms ( 34 ). In this review, following search terms for this kind of cells were adopted: “adipose-derived stromal cells”, “adipose-derived stem cells”, “adipose-derived stromal/stem cells”, “ASCs” and “ADSCs”, and having no limitation to the human or animal species.

3 Monolayer culture techniques

In the 1960s, Rodbell and Jones pioneered the initial method of isolating cells from adipose tissue ( 35 – 37 ). The researchers isolated stromal vascular fraction (SVF) from rat fat pads, which contained heterogeneous cells. In the final step, adherent plastic cells within the SVF were selected and enriched for “preadipocytes”. In 2001, ZUK et al. obtained a fibroblast-like cell population or a processed lipoaspirate from human lipoaspirate. They determined these cells could differentiate into adipogenic, osteogenic, chondrogenic, and myogenic cells in vitro, which opened up new avenues for MSC research ( 38 ). The isolation and culture process of ASCs is shown in Figure 1 .

Figure 1 The isolation process of ASCs. The cells showed are isolated from rat’s inguinal adipose tissue. Scale bar, 200μm. SVF, stromal vascular fraction; ASCs, adipose-derived stromal/stem cells.

The characterization of ASCs involves fulfilling specific criteria related to cellular morphology ( 39 , 40 ), immune-phenotypic ( 10 ), and differentiation capacity ( 10 , 31 ). As high quality of cells is the prerequisite for their application, various factors that may influence their biological functions during culture have been proposed ( Figure 2 ).

Figure 2 Influential factors on biological functions of ASCs during culture. Many aspects are reported to influence ASC culture and their biological functions. These can broadly be divided into the sources of tissues and cells, techniques of isolation, culture and cryopreservation. WAT, white adipose tissue; BAT, brown adipose tissue; FBS, fetal bovine serum.

3.1 Tissue and cell sources

3.1.1 health conditions of donors.

Cells can be obtained from healthy donors or individuals with varying degrees of diabetes, obesity, and other chronic diseases. The use of autologous and allogeneic ASCs should be carefully considered. Autologous cells have advantages in terms of histocompatibility and infectious concerns ( 41 ), but their functionality may be compromised in an unhealthy environment. ASCs derived from diabetic donors have shown reduced proliferation ability and paracrine activity compared to autologous ASCs from healthy individuals ( 42 – 44 ), but they still hold potential in cell therapies ( 45 – 47 ). Additionally, Obesity has an adverse impact on ASCs, resulting in defective functionalities and properties ( 48 ). ASCs from individuals with obesity exhibit decreased telomerase activity and telomere length ( 49 ). There are no significant differences observed in ASCs between oncological patients and healthy subjects ( 50 , 51 ). However, ASCs from donors exposed to radiotherapy and chemotherapy exhibit altered cell migration, proliferation, and differentiation capacity ( 48 ). The outcomes are also correlated with other demographics, such as age, gender, and ethnicity ( 52 ).

3.1.2 Types of adipose tissue

White adipose tissue (WAT) mainly exists in two types: subcutaneous and visceral adipose tissue. ASCs obtained from subcutaneous (S-ASCs) and visceral adipose tissue (V-ASCs) share similar cell viability and surface markers but differ in motility, secretory function, and expression of stemness-related genes ( 53 ). However, S-ASCs have a greater differentiation capacity to adipogenic and osteogenic cells, and V-ASCs proliferate slower, require stronger stimulation for differentiation ( 54 ), and secrete higher levels of inflammatory cytokine such as interleukin (IL)-6, IL-8 and tumor necrosis factor (TNF)-α ( 55 ). Wada et al. ( 56 ). also found that V-ASCs and S-ASCs release inflammatory and angiogenesis cytokines differently. Moreover, ASCs in the superficial layer, located closer to the dermis, exhibited hyperplastic and angiogenic capacities, while ASCs in the deep layer were characterized by inflammatory properties similar to V-ASCs ( 27 ).

Furthermore, studies have shown the presence of ASCs derived from brown adipose tissue (BAT) ( 57 , 58 ). The characteristics of ASCs derived from BAT differ from those of WAT, particularly, the expression of myogenic factor 5 (Myf5) and myogenic origin. In these cells, gene expression profiles are unique, particularly the higher expression of genes associated with BAT including uncoupling protein-1 (UCP1), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), PR domain containing 16 (PRDM16), and CAMP responsive element binding protein one (CREB1). Therefore, the tissue and cell sources should be considered for further application.

3.2 Isolation and culture

3.2.1 collagenase digestion.

The first crucial step in obtaining cells from adipose tissue is cell isolation. Currently collagenase digestion remains the most common method to obtain cells due to its simplicity and high cell purity ( 41 , 48 ). However, the use of xenogeneic collagenase may lead to pathogen transmission and immune response in vivo. To be considered safe, the development of clinical grade digestive products is crucial for the isolation of ASCs. Carvalho et al. demonstrated that several alternative enzyme products, including Collagenase NB 4 Standard Grade (NB4) [Serva], Collagenase Type 1 (CLS1) [Worthington], Collagenase (Animal Origin Free)-A (CLSAFA) [Worthington], and Liberase [Roche], were equally effective as research-grade products ( 59 ). Kølle et al. implemented a clinical trial using cells which were isolated by clinical collagenase NB 6 ( 60 ).

3.2.2 Serum deprivation

Fetal bovine serum (FBS) is another important consideration for ASC culture and application, similar to xenogeneic collagenases. The available studies showed that human ASCs (hASCs) maintain their stemness in serum-deprived medium ( 61 ). In the absence of FBS for 48 hours, hASCs showed reduced metabolism and proliferation, but maintained the expression of crucial surface markers, without undergoing apoptosis or necrosis ( 51 ). Human ASCs cultured in STK2 (a chemically-defined serum-free medium) exhibited enhanced proliferation, elevated expression of MSC surface markers, and diminished cell aging compared to those cultivated in media supplemented with FBS ( 62 ). According to these observations, FBS deprivation does not cause impacts that would prevent cellular clinical application.

Other alternative supplements have been investigated as potential substitutes for FBS. Human platelet lysates (HPLs) could serve as a superior supplement. They were found to augment the proliferative capacity of hASCs in comparison to FBS, while simultaneously preserving their untransformed state and differentiation ability ( 63 , 64 ). Kocaoemer et al. observed that hASCs cultured in medium supplemented with either thrombin-activated platelet rich plasma (tPRP) or pooled human serum (HS) exhibited similar properties, although a reduction in adhesion was observed in cells cultured in tPRP-supplemented medium ( 65 ). According to the whole genome gene analysis, 90 genes were significantly expressed more in hASCs cultured in FBS-supplemented medium ( 66 ).

3.2.3 Oxygen concentration

As the oxygen concentration of adipose tissue in vivo is 2%-8%, ASCs exist in a relatively low-oxygen microenvironment ( 67 , 68 ). However, most ASCs are cultured under normal oxygen conditions (21% oxygen concentration) in vitro. Human subcutaneous ASCs cultured in hypoxic conditions in vitro exhibited increased proliferation rates and secretion of growth factors ( 69 ). Tirza et al. discovered weakened proliferation ability, increased accumulation of reactive oxygen species (ROS), and genetic instability of rat visceral ASCs cultured under normal oxygen experienced, which could be improved by lowering the culture temperature ( 67 ).

3.2.4 Cell cryopreservation

Despite the diminished cell viability and lower colony-forming-unit percentages observed in cells derived from cryopreserved lipoaspirate compared to fresh lipoaspirate-derived cells, the viable cells that remained exhibited preserved adhesive and proliferative properties ( 70 ), which could counteract the negative effect with continued cell growth ( 71 ). After prolonged cryopreservation at 70°C, the number of viable cells decreased as well as their viability ( 71 ). A cryopreservation medium containing HS, HS albumin, or knockout serum replacement did not affect the gene expression, differentiation ability, and immunophenotype of hASCs for a duration of 3-4 freeze-thaw cycles, but significantly reduced the proliferation. Thus, it has been recommended that cells for clinical application should not undergo more than two freeze-thaw cycles ( 72 ).

In summary, isolation and culture methods can affect ASCs properties, therefore, there is still a need to look for appropriate culture protocol that will provide the right number and characteristics of ASCs without affecting their therapeutic potential for clinical application.

3.3 Cellular senescence and potential interventions

Cellular senescence, also called aging, has always been an obstacle to the development of MSC therapy. Some studies confirmed the stability of ASCs during a certain period (usually up to the sixth or seventh passage) ( 51 , 73 , 74 ). However, Yin et al. found that hASCs rapidly underwent replicative senescence and lost stem cell properties over 21 days by current 2D culture ( 75 ). During long-term culture, senescent cells experience a cessation in proliferation, and exhibit distinct morphological and physiological features, including enlarged nuclear and cytoplasmic volumes, heightened β-galactosidase enzyme activity, decreased expression of β cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1), telomere shortening and accumulation of ROS ( 76 , 77 ). The stability and safety of ASCs should be considered in application, thus, many research efforts have been enhanced to address the problem of cellular aging.

Immortalization techniques have been shown to overcome senescence in primary cells ( 78 ). Kang et al. discovered that ectopic expression of telomerase reverse transcriptase (TERT) in non-human primates ASCs enabled cells to maintain proliferative potential and multipotent differentiation ability ( 79 ). Tchkonia et al. generated preadipocyte strains from single abdominal subcutaneous, mesenteric and omental human preadipocytes through stable expression of human TERT (hTERT). These strains were capable of repeated subculturing and maintained the capacity for differentiation, as well as the specific dynamic characteristics of fat depot cells ( 80 ). Wolbank et al. found hTERT overexpression generated ASC lines (ASCs hTERT ) exhibited continuous growth and showed minimal changes in morphology, surface marker profile, karyotype, immunosuppressive capacity and differentiation potential ( 81 ). Similarly, Shamsi and Tseng developed protocols for immortalizing brown and white preadipocytes ( 82 ). Furthermore, researchers have cultured ASCs with TERT expression to conduct further researches in regenerative medicine and other medical fields ( 79 , 83 – 85 ).

Furthermore, Tátrai et al. found that human ASCs hTERT and ASCs generated by the co-transduction of hTERT and Bmi-1 retained MSC features and did not senesce, whereas ASCs generated by the overexpression of Bmi-1 exhibited limited replicative potential. Notably, a subpopulation of ASCs hTERT also acquired aberrant karyotype and showed signs of transformation after long-term culture ( 86 ).

However, Balducci et al. found that hTERT alone failed to immortalize hASCs. Moreover, hASCs that were co-transduced with hTERT and human papillomavirus (HPV)-E6/E7 were successfully immortalized and could secrete significant amount of hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF), albeit with reduced differentiation properties and some chromosomal aberrations ( 87 ). Darimont et al. demonstrated that co-transduction of hTERT and HPV-E7 enabled human preadipocytes to extend their lifespan and maintain their capacity for differentiation ( 88 ).

The overexpression of simian virus 40 large T antigen (SV40T) has been widely employed as a strategy to overcome replicative senescence in human primary cells. However, it was found that the adipogenic differentiation process was blocked by SV40T expression in 3T3-F442A cells ( 89 ). Human ASC lines operated by co-transduction of hTERT and SV40T underwent chromosome aberration, deviated from the normal MSC phenotype, and lose the ability of differentiate ( 86 , 87 ).

Although there were variations in results across different studies, it is generally established that cell immortalization can be achieved through gene editing technology ( Table 1 ). Notably, the possibility of karyotype variation should be taken into consideration in these immortalized cells constructed by gene editing technology.

Table 1 Immortalization of ASCs through gene editing technology.

4 3D culture techniques

Significantly, advancements in stem cell and 3D culture technologies have enabled the creation of cellular models that accurately mimic the histological, molecular, and physiological characteristics of tissues and organs ( 29 ). The formation of 3D cultures relies on the self-organization and differentiation of cells, as well as signaling cues from the extracellular matrix (ECM) and conditioned media ( 90 ).

4.1 Cell types

3D cultures are typically self-assembled in vitro 3D structures derived from primary tissues or various types of stem cells, including MSCs, iPSCs, and ESCs. Various cell types exhibit distinct developmental pathways, underscoring the importance of selecting an appropriate initial cell population for the successful establishment of organoid cultures. 3D culture of ESCs has not been a priority due to their ethical concerns. 3D culture models derived from MSCs have been shown to highly recapitulate the homeostasis and regenerative capacity of the tissue of origin ( 91 ). Conversely, models derived from iPSCs often hardly recapitulate the adult tissue stage, instead resembling the fetal tissue stage ( 92 , 93 ). 3D culture models derived from ASCs are generated without genetic modification by transcription factors, unlike those derived from iPSCs ( 94 ). Moreover, ASC exhibit immune privileged properties, and accordingly show excellent safety for allogeneic transplantation in multiple human clinical trials ( 4 ). Therefore, ASCs is a cell type with great potential and advantages in 3D culture technology.

4.2 Effects of culture techniques on cellular properties

Despite numerous studies, there is no standardized method for 3D ASC culture. It is necessary to comprehend the impact of different 3D culture techniques on cellular properties in contrast to traditional 2D culture.

4.2.1 Cell viability and stemness

The stemness properties of MSCs are retained in the in vivo microenvironment, which includes soluble growth factors, cell-cell interactions and cell-matrix interactions ( 95 , 96 ). Increasing evidence has indicated that the cellular microenvironment significantly influences stemness properties ( 95 , 97 ). In comparison to conventional monolayer cultures, 3D cultured methods provide a cellular niche that more closely resembles the in vivo microenvironment ( 98 ).

Existing techniques for ASC culture can be categorized into scaffold-free and scaffold systems ( 26 , 99 ). The conventional scaffold-free culture techniques, such as the use of low adhesion plates, hanging drops, and spinner flasks, have been shown to impact the viability and stemness of ASCs.

Low adhesion plate culture method involves the formation of spheroids by suspending cells on a surface with low adhesion properties. Guo et al. successfully generated 3D spheroids using non-adhesive agarose Petri dishes. This method was found to overcome poor post thaw cells and improve the viability and neural differentiation potential of hASCs ( 100 ). Similarly, Coyle et al. conducted a study examining hASC spheroids with various sizes and demonstrated the enhanced viability of spheroids was achieved through anaerobic glycolysis in conditions of increased glucose availability and decreased oxygen levels ( 101 ). Di Stefano et al. conducted a comparative analysis of hASCs cultured in ultralow culture flasks and hASCs with 2D primary cultures. Their study identified distinct molecular expression patterns of genes associated with stemness, as well as genes related to anti-aging, oxidative stress, and telomeres maintenance of hASCs ( 102 ). Rybkowska et al. conducted a study in which 3D hASC spheroids were cultured using antiadhesive plates. However, they observed that the spheroids exhibited slightly lower viability, reduced proliferation rates, but higher expression of stemness-related transcriptional factors compared to cells cultured in monolayer. Additionally, the 3D culture resulted in increased mitochondrial DNA content, oxygen consumption rate, and extracellular acidification rate. Elevated levels of ROS and decreased intracellular lactic acid levels were also detected ( 103 ).

The hanging drop method technique capitalizes on the intrinsic tendency of cells to self-assemble into three-dimensional aggregates needless of scaffolding. A drop is formed within an inverted plate and held in place due to surface tension. Jin et al. utilized the hanging-drop technique to produce hASC microtissues in a smooth muscle inductive medium supplemented with human transforming growth factor β1, and subsequently bioprinted these induced microtissues onto a 3D framework. The microtissues retained their phenotypic characteristics post-bioprinting. Cell viability and proliferation within the 3D microtissues were consistently superior in comparison to the traditional single-cell bioprinting approach ( 104 ).

The spinner flask facilitates the generation of fluid flow, which discourages cellular adhesion and facilitates cellular aggregation. Bangh et al. placed hASC spheroids in spinner flasks under 1% oxygen. The spheroids exhibited faster growth rates compared to monolayer cultures. Additionally, they observed an upregulation of survival factors in response to the spheroid size ( 105 ).

Another commonly employed approach involves seeding stem cells into scaffolds that mimic the ECM of native tissues, which can be fabricated using biologically derived or synthetic materials. Natural scaffolds consist predominantly of collagen, fibrin, gelatin, vitronectin, laminin, alginate, hyaluronic acid (HA), or decellularized materials, while synthetic scaffolds may consist of materials such as polyesters, polyethers, polyethylene glycol, and polylactic acid (PLLA) ( 106 ). Several studies have investigated the use of different hydrogels to create ASC spheroids, utilizing commonly used materials in tissue engineering such as HA and chitosan, resulting in enhanced stemness gene expression compared to traditional adhesion plate cultures ( 107 – 110 ). A poly(ethylene glycol) (PEG) hydrogel microwell pattern was fabricated on a poly(N-isopropylacrylamide) hydrogel substrate to regulate the size of spheroids. The viability of hASC spheroids exceeded 97.5% ( 111 ).

Based on mechanical structure or new systems, various novel techniques were devised to create 3D ASC structures with enhanced viability, increased stemness, and enhanced differentiation capabilities, such as the following: A switchable water-adhesive, super-hydrophobic nanowire surface ( 112 ); microgravity bioreactors ( 113 ); microwell plates employed with gelatin microparticles ( 114 ); microfabricated porous tissue strands (pTSs) ( 115 ); a method defined “all-in-one platform” with hydrogels with an embossed surface (HES) ( 116 ); gelatin hydrogels with microbial transglutaminase (mTG) ( 117 ); and TeSR-E8 medium (a highly chemically defined medium) in conventional tissue culture polystyrene dishes ( 118 ). Furthermore, Labriola et al. utilized polymer-based, cell mimicking microparticles (CMMPs) to deliver distinct, stable mechanical cues to hASCs in 3D spheroid culture. Mechanically tuned CMMPs controlled whole-spheroid mechanical phenotype and stability but minimally affected differentiation response ( 119 ).

Based on the findings of these studies, it is evident that the majority of research indicates that 3D culture enhances cell viability, stemness, proliferation rate, and metabolic functions, with only a few exceptions showing a decrease in cell viability.

4.2.2 Differentiation ability

Multilineage differentiation potential of ASCs towards both mesenchymal and non-mesenchymal lineage cells have been reported, particularly towards adipogenic, chondrogenic, and osteogenic lineages, which can be facilitated by the introduction of lineage-specific factors ( 120 ).

Adipogenic differentiation: Decellularized adipose tissue (DAT) based hydrogels have been demonstrated to closely replicate the native ECM environment, effectively inducing adipogenic differentiation and promoting the proliferation of hASCs ( 121 , 122 ). In a study by Zhang et al., hASC spheroids cultured in a microgravity bioreactor exhibited enhanced stemness properties and adipogenic differentiation potential compared to monolayer culture ( 113 ). Hoefner et al. cultured hASC spheroids in growth cell media under agitation at 50 revolutions per minute. After a brief 2-day induction period for adipogenic lineages, it was observed that ASC spheroids exhibited enhanced differentiation capacity within their own ECM when compared to traditional 2D cultures ( 123 ). These findings suggest that utilizing 3D ASC culture may be a promising approach for adipose tissue engineering applications.

However, Rumiński et al. reported that hASC spheroids seeded in 96-well sterile round-bottom culture plates and subjected to gentle rotation on a rotary shaker displayed reduced adipocyte differentiation ( 124 ). The elastin-like polypeptide (ELP)-polyethyleneimine (PEI) coated surface was demonstrated a suitable cell culture material ( 125 ). However, the study conducted by Turner et al. revealed that triglyceride accumulation was less pronounced in hASC spheroids seeded on ELP-PEI coated surfaces compared to 3T3-L1 adipocytes, correlated with smaller average spheroids, suggesting a relatively slower differentiation process ( 126 ).

Chondrogenic differentiation: Yoon et al. employed the spinner flask method to illustrate that 3D hASC spheroids exhibit enhanced chondrogenic capabilities when cultured in a specific differentiation medium as opposed to monolayer culture ( 127 ). Tsai et al. employed mTG, an enzyme with high specificity across a broad temperature range, to crosslink gelatin. The evaluation of differentiation potential revealed that hASC spheroids within the 3D gelatin/mTG hydrogel demonstrated heightened activity, particularly in adipogenesis and chondrogenesis, in comparison to the cell suspension group ( 117 ). Furthermore, when comparing hASC spheroids cultured using microwell techniques to ASCs cultured in a 2D monolayer, it was observed that cell survival and chondrogenic potential were enhanced, while apoptosis was diminished. Injecting hASC spheroids exerted enhanced regenerative capabilities for articular cartilage and effectively halted the advancement of surgically induced osteoarthritis through the paracrine mechanism of action, when compared to ASCs in single-cell suspension ( 128 ).

Osteogenic differentiation: Gurumurthy et al. illustrated that 3D hASCs cultivated on ELP-PEI scaffolds exhibited a heightened propensity for differentiation towards the osteogenic lineage in comparison to 2D cultures ( 129 ). Human ASCs were cultured in 3D systems devoid of bioactive material components: spheroids and polystyrene scaffolds. Alkaline phosphatase activity, a marker of early osteogenesis, exhibited increased levels in ASC spheroids and ASC-seeded scaffolds in comparison to 2D cultures. The expression of the osteoblast marker, including Runt-related transcription factor 2, and osterix and integrin binding sialoprotein was significantly up-regulated in spheroids compared to polystyrene scaffolds and 2D culture ( 124 ). Kim et al. conducted a study to evaluate the osteogenic potential of hASCs in 2D and 3D culture environments. Through comprehensive analysis of transcriptome sequencing data, they identified an upregulation of genes associated with skeletal development, bone formation, and bone remodeling processes in hASCs cultured in concave microwells ( 130 ).

Differentiation into other lineages: Cheng et al. utilized chitosan films to form hASC spheroids, which, when cultured in appropriate induction media, exhibited enhanced differentiation capabilities, including differentiation into neuron and hepatocyte-like cells ( 131 ). Guo et al. observed an increased capacity for neural differentiation in 3D hASC spheroids cultured in agarose 3D Petri dishes ( 100 ). Amirpour et al. employed a defined neural induction medium with small molecules to directly differentiate hASCs into anterior neuroectodermal cells using hanging drop protocols ( 132 ). Additionally, Salehi et al. conducted a comparison between two differentiation protocols for the generation of retinal precursor-like cells in vitro: hASCs monolayer culture and hanging drop culture with a defined medium. The study indicated that the hanging drop method led to an enhanced yield of retinal precursor differentiation, resulting in precursor-like cells that exhibited responsiveness to the glutamate neurotransmitter ( 133 ). Moreover, the hanging drop method was found to enhance the efficiency of hASC smooth muscle differentiation and improve cell viability within a 3D bioprinted structure ( 104 ). Bagheri-Hosseinabadi et al. observed a higher rate of cardiomyogenic differentiation in hASCs cultured in a 3D hanging drop system with 5-azacytidine compared to the 2D culture ( 134 ).

These findings suggest that the 3D environment may offer enhanced stimuli for the differentiation of ASCs into various lineages. These results have implications for the development of protocols for preparing ASCs for use in clinical studies focused on regeneration.

4.2.3 Paracrine secretion

The paracrine secretion of cytokines such as angiogenic factors, adipokines, neurotrophic factors, and interleukin plays a crucial role in the therapeutic application of ASCs by promoting tissue regeneration and repair ( 120 ).

3D cultured ASCs possess distinct and inherent characteristics independent of the method of formation. The size of 3D cultured ASCs is a critical factor, as larger cells exhibit higher levels of hypoxic factors that stimulate angiogenesis and antiapoptotic gene expression ( 26 ). small spheroids of average spherical shape were generated in 96-well plates. The 3D condition of the hASCs was found to be correlated with elevated levels of VEGF-A and IL-8 expressions in relation to wound healing ( 135 ). Kim et al. introduced HES as a comprehensive platform capable of facilitating the rapid formation and cultivation of a substantial quantity of size-adjustable 3D hASC spheroids. Notably, HES-derived spheroids exhibited a higher VEGF secretion compared to spheroids cultured on a commercially available low-attachment culture plate. Utilizing these advantages, HES-based spheroids were employed for 3D bioprinting, resulting in enhanced retention and VEGF secretion within the 3D-printed construct compared to a similar structure containing single cell suspension ( 116 ). Yu et al. utilized agarose microwells to seed hASCs, generating uniform cell spheroids with adjustable size, and stimulated ECM deposition through the use of ascorbic acid 2-phosphate to form ASC sheets. Transcriptome sequencing analysis indicated upregulation of angiogenesis-related genes in ASC spheroids compared to monolayer ASCs. The study illustrated the stimulatory impact of spheroid formation on ASCs towards endothelial lineage by observing increased expression of cluster of differentiation (CD) 31, which persisted following the seeding of ASC spheroids on cell sheets. Furthermore, compared to ASC sheets, ASC spheroid sheets exhibited heightened expression of VEGF and HGF, and the conditioned medium from ASC spheroid sheets significantly promoted tube formation of endothelial cells in vitro ( 136 ).

Seo et al. innovatively created a switchable water-adhesive, super-hydrophobic nanowire surface to enhance cell-cell and cell-matrix interaction, leading to improved cell viability and paracrine secretion of VEGF in hASC spheroids. The size of hASC spheroids can be easily manipulated on this surface. Accordingly, the spheroids generated on this surface demonstrate significantly heightened angiogenic effectiveness in comparison to spheroids produced through traditional methods such as spinner flask suspension culture and hanging drop culture on a petri dish ( 112 ). The successful establishment of a 3D co-culture model utilizing HA gel and a 10:1 ratio of late-passage hASCs and endothelial colony-forming cells resulted in increased secretion of cytokines, including HGF, VEGF, and epidermal growth factor (EGF), compared to single-cell 3D culture or monolayer culture ( 109 ). These findings suggest potential applications of 3D strategies in angiogenesis and regeneration therapies.

Furthermore, Zhang et al. utilized a low-adhesion cell culture plate to generate rat ASCs (rASCs) into microtissues in vitro. They employed grafts composed of microtissues and polycaprolactone nerve conduit for the purpose of repairing sciatic nerve defects in rats. Their study revealed that microtissues promote the secretion of nerve regeneration-related cytokines, including brain-derived neurotrophic factor, and nerve growth factor, the angiogenic factor such as VEGF, as well as anti-inflammatory cytokines such as IL-4, IL-10, and IL-13. This secretion ultimately facilitated the growth of axons when compared to an equivalent number of cells cultured in a 2D manner ( 137 ). Zhou et al. utilized a hanging drop method to generated murine ASCs-based microtissues, which were subsequently injected into streptozotocin (STZ)-induced diabetic rats for the treatment of erectile dysfunction. The findings demonstrated elevated expression of VEGF, nerve growth factor, and TNF-stimulated gene-6 within the microtissues, indicating neuroprotective and anti-inflammatory properties ( 138 ).

Overall, the use of specific culture media and 3D cultured techniques can enhance the differentiation potential and paracrine secretion of ASCs. Hence, it is imperative to carefully deliberate on the selection and refinement of techniques for producing 3D cultured ASCs, as they have the potential to impact the characteristics of cells.

5 The potential of 3D cultured ASCs for diabetic therapy

Diabetes as a multi-organ disease, is a significant cause of increased morbidity and mortality worldwide. In the treatment of diabetes and its complications, ASCs have been used due to their inherent attributes such as self-renewal capacity, differentiation potential, homing mechanism and immunosuppressive property ( 11 , 21 , 139 ). Currently, the clinical trials of ASCs for treating diabetes and its associated complications, including the diabetic foot ulcer (DFU), diabetic critical limb ischemia, and diabetic nephropathies, are still in the preliminary research stage ( Table 2 ). There is a lack of agreement regarding the optimal method of administration to achieve enhanced therapeutic outcomes. Potential routes of administration include intravascular injection, local tissue injection, and thymus injection. In diabetic patients, the most commonly used administration routes are intraportal injection and intravenous infusion ( 140 , 141 , 143 , 144 ). For diabetic angiopathy conditions like DFU, common delivery methods include local injection of ASCs and direct application of 3D ASC grafts onto the wound site ( 145 – 147 ).

Table 2 Completed and ongoing clinical trials of ASCs in diabetes and its complications.

Unfortunately, their efficacy is primarily impeded by the limited expansion and survival of transplanted stem cells and their inability for proper functional integration in response to the physical environment ( 21 , 149 ). Moreover, only a fraction of MSCs successfully home to the pancreas and express insulin ( 150 ). 3D culture technology provides an opportunity to fill this knowledge gap. While there is a scarcity of research on the clinical application of 3D cultured ASCs, findings from animal and cellular studies suggest the potential benefits and advantages of 3D cultured ASCs in the treatment of diabetes ( Table 3 ).

Table 3 The characteristics of 3D cultured ASCs in diabetes and diabetic complications compared to monolayer cells.

5.1 Promotion of insulin production

Type 2 diabetes mellitus (T2DM) is the most common type of diabetes, characterized by two interrelated metabolic defects: insulin resistance and pancreatic islet β-cell dysfunction. The development of T2DM is influenced by a complex interplay of genetic, environmental, emotional, and behavioral factors ( 151 ). Individuals with T2DM typically exhibit insulin resistance and gradual β-cell deterioration, resulting in insufficient insulin secretion, and consequent hyperglycemia and elevated free fatty acid levels. The resulting glucotoxicity and lipotoxicity exacerbate the dysfunction of β-cells to secrete insulin in response to hyperglycemia or oral hypoglycemic agents ( 16 ).

Type 1 diabetes mellitus (T1DM) is a chronic disease characterized by insulin deficiency resulting from autoimmune destruction of pancreatic islet β-cells, ultimately leading to hyperglycemia. Although the mechanism of T1DM in still not completely understood, it is believed to involve abnormalities in multiple immune cells, including T cells, B cells, regulatory T cells, monocytes and macrophages, and dendritic cells ( 152 ).

Given their similar outcome of pancreatic islet β-cell dysfunctions, the cell therapy as a potential strategy has attracted increased research attention. However, the transplantation of functional β-cells as a therapeutic strategy is impeded by the significant challenge of generating an adequate quantity of β-cells ex vivo and subsequently maintaining their viability post-transplantation. β-cells are susceptible to hypoxia and are prone to rapid apoptosis or damage as a result of the host immune response ( 153 ). Notably, the use of 3D cultured ASCs significantly promotes the construction and transplantation of islets and promote the insulin production.

Firstly, 3D cultured ASCs are capable to differentiate into insulin-producing cells (IPCs) to promote insulin production. For example, Khorsandi et al. found that the collagen/HA scaffold could enhance the differentiation of IPCs from rASCs. Compared to the 2D culture, the insulin release from 3D ASCs-derived IPCs showed up-regulation when exposed to a high glucose medium. The percentage of insulin-positive cells in 3D culture showed an approximately 4-fold increase compared to the 2D cultured cells ( 154 ). Ikemoto et al. developed a human recombinant peptide petaloid μ-piece 3D culture method to generate IPCs from hASCs. Following transplantation of 96 IPCs under the kidney capsule or intra-mesentery in STZ-induced diabetic nude mice, the hyperglycemic state was restored to normoglycemia ( 155 ). Ohta et al. found that blood glucose levels of STZ-induced diabetic nude mice were normalized after transplantation of 3D-cultured IPCs ( 156 ).

Secondly, the immunomodulatory action of 3D cultured ASCs can improve the micro-environment of islets. Abadpour et al. developed 3D-printed bioactive scaffolds containing islets and hASCs by combining alginate and nano-fibrillated cellulose bioink. Bioink diffusion properties were demonstrated, as well as benefits of hASCs for glucose sensing, insulin secretion, islet viability, and the reduction of pro-inflammatory cytokines, including growth-regulated protein-α and interferon gamma-induced protein-10 ( 157 ).

Furthermore, the 3D culture methods can also facilitate the survival of pancreatic islets and increase the functionality of grafts before transplantation. Jun et al. introduced a method of transplantation by co-culturing single primary islet cells with rASCs in concave microwells. These spheroids exhibited distinct ultrastructural morphologies, increased viability, and enhanced insulin secretion compared to mono-cultured islet spheroids, suggesting that ASCs may protect islet cells from damage by releasing anti-apoptotic growth factors. Additionally, the co-encapsulation of islets with additional ASCs within microfibers could further prolong graft survival through the anti-inflammatory properties of ASCs ( 22 ). Wang et al. effectively produced viable and functional heterocellular islet micro-tissues by combining islet cells, human umbilical vein endothelial cells, and hASCs within porcine decellularized ECM. These 3D islet micro-tissues exhibited sustained viability and normal secretory function, as well as heightened drug sensitivity during testing. Additionally, the utilization of 3D islet micro-tissues resulted in improved survival rates and enhanced graft function in murine models of diabetes ( 158 ).

In conclusion, 3D cultured ASC grafts play more significant role of insulin production through differentiating into IPCs, improving the micro-environment of islets, and enhancing survival and functionality.

5.2 Treatment of diabetic foot ulcer

Diabetic complications are mainly caused by high-glucose-induced cellular and molecular impairments and dysfunctions of cardiovascular and neural systems. While monolayer ASCs have demonstrated efficacy in treating a range of diabetic complications ( 139 ), the current studies about treatment of 3D ASCs are mainly focused on the DFU.

The diabetic foot ulcer, considered among the most severe types of diabetic wounds, has significant challenges to healing due to diabetic neuropathy, reduced blood flow, and infections ( 159 ). Non-healing ulcers may progress to gangrene, requiring foot amputations.

The normal wound healing process is characterized by four stages: hemostasis, inflammation, proliferation, and remodeling. In the hemostasis stage, vasoconstriction, platelet aggregation, and recruitment of circulating coagulation factors occur. The inflammation stage involves the gathering of inflammatory cells that secrete inflammatory factors like matrix metalloproteinase (MMP) and neutrophil extracellular traps (NETs). During the proliferation stage, the inflammation diminishes, and skin cells such as keratinocytes secrete EGF, proliferate, and migrate to the wound bed. During the process of tissue remodeling, new tissue is restructured and deposited via ECM and neovascularization, facilitated by fibroblasts secreting FGF and vascular endothelial cells secreting VEGF ( 160 – 162 ).

In diabetic wounds, tissue ischemia, hypoxia, and a high glucose microenvironment disrupt the normal progression of these healing stages, leading to delayed or non-healing of wounds and various clinical complications ( 163 ). Currently, DFUs are treated with vascular intervention therapy, drugs and other non-surgical therapies, such as dressing adjuvant therapy, hyperbaric oxygen therapy, hyperthermia and growth factor therapy ( 164 ). However, the efficacy of these approaches remains limited ( 159 ). Therefore, future research endeavors are anticipated to concentrate on more effective treatment strategies, with a particular emphasis on advancing stem cell-based therapies.

ASCs exhibit significant promise in the treatment of diabetic foot ulcers. Basically, the effects of ASCs rely on their promotion of immunomodulation, neovascularization and fibro synthesis ( 165 , 166 ). The routes of delivery of ASCs into the wound vary between direct injection (such as intradermal injection around the wound, intra-fascial, and intramuscular injection), topical gel treatment, engineered skin graft sheet, and with scaffolds. The survival rate and potency of expansion of ASCs in wound bed are limited in traditional injection. Therefore, scaffolds cell delivery systems are necessary which offer optimal environments for cell adhesion, proliferation, and differentiation ( 167 , 168 ).

A common solution involves seeding cells into hydrogels. Zeng et al. proposed that gelatin microcryogels (GMs) presented a novel method of cell delivery that could not only enhance wound bed healing but also directly influence the basal layer of the wound. They demonstrated that GMs provided an enhanced microenvironment for inducing endothelial cell differentiation of hASCs, thereby offering potential in vivo applications for angiogenic regeneration. Additionally, they demonstrated the priming effects of GMs on the upregulation of stemness genes and improved secretion of crucial growth factors in hASCs for wound healing, such as VEGF, HGF, basic fibroblast growth factor (bFGF), and platelet-derived growth factor BB (PDGFbb) ( 169 ). Feng et al. examined the therapeutic potential of hASCs cultured as micro-spheroids in the HA gel. Diabetic ulcers in mice with hASC spheroids resulted in accelerated wound epithelialization and increased dermal thickness, surpassing the outcomes observed with vehicle alone or monolayer-cultured ASCs ( 170 ). An injectable hydrogel system based on PEG and gelatin was examined for delivering hASCs into diabetic wounds. The stemness-linked transcription factor expression of hASCs was preserved in vitro and cell retention was significantly enhanced in vivo by this gel. In diabetic mice, this ASC-hydrogel treatment reduced inflammatory cell infiltration, enhanced neovascularization, and sped up wound closure ( 23 ).

There are also other bioengineering approaches for constructing 3D cultured ASCs. For example, Tyeb et al. introduced a combinatorial method involving the utilization of gelatin-sericin (GS) scaffolds coated with laminin (GSL). GS scaffolds provided enhanced protection against free radical-induced damage compared to gelatin scaffolds and consequently improved cell viability and metabolic function. The utilization of rASCs loaded onto GSL scaffolds resulted in enhanced regeneration, collagen remodeling, and increased expression of CD31 in diabetic ulcer rat models ( 171 ).

However, the broad use of matrix components aiding in the formation of 3D structures may impose constraints on the clinical applicability owing to the presence of undefined components. The implementation of hASCs formulated as multicellular aggregates without scaffolds also facilitated the healing wounds of diabetic mice. These aggregates exhibited a noteworthy increase in the production of extracellular matrix proteins including tenascin C, collagen VI α3, and fibronectin, as well as the secretion of soluble factors including HGF, MMP-2, and MMP-14 when compared to monolayer culture ( 172 ).

Considering that the main mechanism of cell action involves the paracrine effect, the characterization of components secreted by cells is vital, which indicates that ASCs can also function through their conditioned media. Lee et al. successfully fabricated an alginate-based scaffold using 3D printing and electrospinning techniques, which served as a structure to encapsulate hASC spheroids. This structure not only securely entrapped the spheroids but also facilitated the stable release of factors associated with angiogenesis and wound healing, such as CD31, VEGF, HGF, C-X-C chemokine receptor type 5 (CXCR5), IL-8, and MMP-1. They also demonstrated the role of these factors through a tube-forming assay and found that conditioned media from the spheroid-scaffold group enhanced the formation of capillary-like structures in human umbilical vein endothelial cells when compared to the single cell-scaffold group ( 173 ).

Utilizing diverse 3D culture techniques and materials such as hydrogels, bioactive scaffolds, scaffold-free methods, and conditioned media from 3D cultured cells, ASCs have the potential to facilitate diabetic wound healing by the promotion of immunomodulation, neovascularization and fibro synthesis.

5.3 Modelling tissues and organs

Aside from their application in diabetic therapy through transplantation, 3D cultured ASCs are crucial in the development of in vitro models that mimic the pathophysiology of different tissues and organs linked to diabetes and its associated complications. These models also potentially serve as valuable tools for screening novel therapeutic interventions and minimizing the reliance on animal experimentation.

Adipose tissue is a significant location of insulin resistance in individuals with type 2 diabetes mellitus (T2DM) and is linked to heightened chronic inflammation. The establishment of in vitro models for investigating the pathogenesis of adipose tissue in metabolic diseases would offer significant benefits. Numerous efforts have been made to create 3D adipose cultures utilizing ASCs. For instance, hASCs were cultivated on plates coated with ELP–PEI copolymer, as the PEI component promotes spheroid formation and the ELP component facilitates the attachment of spheroids to the surface. This culture platform enabled the production of functional adipocytes that exhibited a favorable response to fatty acid stimulation ( 126 ). Moreover, Gerlach et al. utilized multicompartment hollow fiber-based bioreactor technology to generate 3D adipose tissue. In vitro, 3D bioreactors allowed greater metabolic activity compared with traditional 2D cultured hASCs and enabled the generation of adipose tissue as long as two months ( 174 ). Yang et al. created a 3D human adipose microtissue engineered within a microfluidic system ( 175 ). Furthermore, culture technologies have been employed in the generation of beige or brown adipose tissue ( 176 – 178 ). As the characterization of ASCs can be influenced by the source of adipose tissue, the availability of such tools presents a wide range of opportunities in vitro studies. By utilizing these models, it becomes feasible to compare relative metabolic responses of adipose depots under different health conditions to metabolic researches.

An ideal and comprehensive adipose tissue models should include all in vivo components, such as adipocytes, connective tissues, veins and nerves. For example, Lau et al. described an adipose micro-physiological system that involved sandwiching human WAT between tissue-engineered sheets of ASCs. The use of ASCs provided a structural ECM framework to encompass and support the mature adipocytes as well as paracrine growth factors ( 179 ). One common approach in the generation of vascularized adipose tissue involves the inclusion of exogenous endothelial cells through co-culture ( 180 – 182 ). The utilization of vascularized adipose models presents a promising avenue for developing novel drugs to treat metabolic diseases by modulation of the adipose vasculature. Moreover, adipose depots could be infiltrated with inflammatory and immune cells during preparation or after differentiation into adipocytes ( 183 ), offering a valuable tool for immune–metabolic research.

Furthermore, through differentiation and secretory capabilities of ASCs, it becomes possible to connect them with micro-physiological systems representing other organs. Despite being in the early stages of development, 3D models simulating organs such as the pancreas, blood vessels, skin, bones, cardiac and skeletal muscles, and nerves ( Table 4 ), exhibit promising potential in mimicking the effects of diabetes and its complications, as well as evaluating the efficacy of cell transplantation therapy.

Table 4 Examples about 3D cultured ASC models of various organs except adipose tissues.

6 Conclusions and future prospects

3D cultured cells have been advantageous in various biomedical fields. This technology is still in the early stages. The isolation, culture, and identification of cells are the basis of 3D culture. Therefore, large-scale manufacturing methods incorporating quality control are necessary for producing cells and 3D cultured transplantations.

Indeed, 2D adherent cell culture of ASCs is still conventionally used for both in vitro and in vivo studies. These cells have been extensively characterized, whereas many factors have not been analyzed on 3D cultured ASCs yet. Additionally, while monolayer ASC cells have shown effects for the treatment of diabetes and its complications in both clinical trials and animal experiments, current research status on 3D cultured ASCs mainly concentrates on T1DM and DFU only. Thus, further research is required to better understand the function and underlying mechanisms of 3D cultured ASC therapy.

The potential side effects of ASCs for tumor development should not be disregarded in studies; however, they may also serve as a potential tool for antitumor therapies. While tumor cells altering the phenotype and function of in vitro cultured ASCs through paracrine mechanisms ( 203 ), ASCs can also serve as a factor that promotes tumor growth ( 203 – 205 ). By contrast, ASC exosomes were shown to possess immunomodulatory properties and can inhibit cancer growth, migration, and colony formation ( 206 ). A strategy for tumor therapy used ASCs which loaded gold nanorod (AuNR)-PEG-poly(ethyleneimine) (APP) and Chlorin e6 (Ce6). Following activation of the APP/Ce6 agents through irradiation, ASCs were shown to play a role in tumor migration, tropism, and exhibit anticancer properties ( 207 ). These findings underscore the importance of exercising caution in the utilization of 3D cultured ASCs, with long-term experiments necessary to assess their safety. Specifically, careful consideration should be given to the potential of 3D culture to induce tumorigenesis while enhancing cell viability and stemness.

Author contributions

YS: Conceptualization, Writing – original draft. XY: Conceptualization, Writing – original draft. JM: Writing – review & editing. WK: Writing – review & editing. XH: Writing – review & editing. JZ: Writing – review & editing. LC: Project administration, Supervision, Writing – review & editing.

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was supported by the National Natural Science Foundation of China (82170822, 82070809, 82300895, and 81900734).

Acknowledgments

The authors thank Figdraw for providing access to create figures.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

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Keywords: adipose-derived stem/stromal cells, monolayer culture, three-dimensional culture, diabetes mellitus, diabetic foot ulcer

Citation: Shi Y, Yang X, Min J, Kong W, Hu X, Zhang J and Chen L (2024) Advancements in culture technology of adipose-derived stromal/stem cells: implications for diabetes and its complications. Front. Endocrinol. 15:1343255. doi: 10.3389/fendo.2024.1343255

Received: 23 November 2023; Accepted: 29 March 2024; Published: 12 April 2024.

Reviewed by:

Copyright © 2024 Shi, Yang, Min, Kong, Hu, Zhang and Chen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) . The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Lulu Chen, [email protected] ; Jiaoyue Zhang, [email protected]

† These authors have contributed equally to this work

Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.

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Stem cell-based therapy for human diseases

Duc m. hoang.

1 Department of Research and Development, Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec Healthcare System, Hanoi, Vietnam

Phuong T. Pham

2 Department of Cellular Therapy, Vinmec High-Tech Center, Vinmec Healthcare System, Hanoi, Vietnam

Trung Q. Bach

Anh t. l. ngo, quyen t. nguyen, trang t. k. phan, giang h. nguyen, phuong t. t. le, van t. hoang, nicholas r. forsyth.

3 Institute for Science & Technology in Medicine, Keele University, Keele, UK

Michael Heke

4 Department of Biology, Stanford University, Stanford, CA USA

Liem Thanh Nguyen

Associated data.

All data generated or analyzed in this study are included in this published article.

Recent advancements in stem cell technology open a new door for patients suffering from diseases and disorders that have yet to be treated. Stem cell-based therapy, including human pluripotent stem cells (hPSCs) and multipotent mesenchymal stem cells (MSCs), has recently emerged as a key player in regenerative medicine. hPSCs are defined as self-renewable cell types conferring the ability to differentiate into various cellular phenotypes of the human body, including three germ layers. MSCs are multipotent progenitor cells possessing self-renewal ability (limited in vitro) and differentiation potential into mesenchymal lineages, according to the International Society for Cell and Gene Therapy (ISCT). This review provides an update on recent clinical applications using either hPSCs or MSCs derived from bone marrow (BM), adipose tissue (AT), or the umbilical cord (UC) for the treatment of human diseases, including neurological disorders, pulmonary dysfunctions, metabolic/endocrine-related diseases, reproductive disorders, skin burns, and cardiovascular conditions. Moreover, we discuss our own clinical trial experiences on targeted therapies using MSCs in a clinical setting, and we propose and discuss the MSC tissue origin concept and how MSC origin may contribute to the role of MSCs in downstream applications, with the ultimate objective of facilitating translational research in regenerative medicine into clinical applications. The mechanisms discussed here support the proposed hypothesis that BM-MSCs are potentially good candidates for brain and spinal cord injury treatment, AT-MSCs are potentially good candidates for reproductive disorder treatment and skin regeneration, and UC-MSCs are potentially good candidates for pulmonary disease and acute respiratory distress syndrome treatment.

Introduction

The successful approval of cancer immunotherapies in the US and mesenchymal stem cell (MSC)-based therapies in Europe have turned the wheel of regenerative medicine to become prominent treatment modalities. 1 – 3 Cell-based therapy, especially stem cells, provides new hope for patients suffering from incurable diseases where treatment approaches focus on management of the disease not treat it. Stem cell-based therapy is an important branch of regenerative medicine with the ultimate goal of enhancing the body repair machinery via stimulation, modulation, and regulation of the endogenous stem cell population and/or replenishing the cell pool toward tissue homeostasis and regeneration. 4 Since the stem cell definition was introduced with their unique properties of self-renewal and differentiation, they have been subjected to numerous basic research and clinical studies and are defined as potential therapeutic agents. As the main agenda of regenerative medicine is related to tissue regeneration and cellular replacement and to achieve these targets, different types of stem cells have been used, including human pluripotent stem cells (hPSCs), multipotent stem cells and progenitor cells. 5 However, the emergence of private and unproven clinics that claim the effectiveness of stem cell therapy as “magic cells” has raised highly publicized concerns about the safety of stem cell therapy. The most notable case involved the injection of a cell population derived from fractionated lipoaspirate into the eyes of three patients diagnosed with macular degeneration, resulting in the loss of vision for these patients. 6 Thus, as regenerative medicine continues to progress and evolve and to clear the myth of the “magic” cells, this review provides a brief overview of stem cell-based therapy for the treatment of human diseases.

Stem cell therapy is a novel therapeutic approach that utilizes the unique properties of stem cells, including self-renewal and differentiation, to regenerate damaged cells and tissues in the human body or replace these cells with new, healthy and fully functional cells by delivering exogenous cells into a patient. 7 Stem cells for cell-based therapy can be of (1) autologous, also known as self-to-self therapy, an approach using the patient’s own cells, and (2) allogeneic sources, which use cells from a healthy donor for the treatment. 8 The term “stem cell” were first used by the eminent German biologist Ernst Haeckel to describe the properties of fertilized egg to give rise to all cells of the organism in 1868. 9 The history of stem cell therapy started in 1888, when the definition of stem cell was first coined by two German zoologists Theodor Heinrich Boveri and Valentin Haecker, 9 who set out to identify the distinct cell population in the embryo capable of differentiating to more specialized cells (Fig. ​ (Fig.1a). 1a ). In 1902, studies carried out by the histologist Franz Ernst Christian Neumann, who was working on bone marrow research, and Alexander Alexandrowitsch Maximov demonstrated the presence of common progenitor cells that give rise to mature blood cells, a process also known as haematopoiesis. 10 From this study, Maximov proposed the concept of polyblasts, which later were named stem cells based on their proliferation and differentiation by Ernst Haeckel. 11 Maximov described a hematopoietic population presented in the bone marrow. In 1939, the first case report described the transplantation of human bone marrow for a patient diagnosed with aplastic anemia. Twenty years later, in 1958, the first stem cell transplantation was performed by the French oncologist George Mathe to treat six nuclear researchers who were accidentally exposed to radioactive substances using bone marrow transplantation. 12 Another study by George Mathe in 1963 shed light on the scientific community, as he successfully conducted bone marrow transplantation in a patient with leukemia. The first allogeneic hematopoietic stem cell transplantation (HSCT) was pioneered by Dr. E. Donnall Thomas in 1957. 13 In this initial study, all six patients died, and only two patients showed evidence of transient engraftment due to the unknown quantities and potential hazards of bone marrow transplantation at that time. In 1969, Dr. E. Donnall Thomas conducted the first bone marrow transplantation in the US, although the success of the allogeneic treatment remained exclusive. In 1972, the year marked the discovery of cyclosporine (the immune suppressive drug), 14 the first successes of allogeneic transplantation for aplastic anemia and acute myeloid leukemia were reported in a 16-year-old girl. 15 From the 1960s to the 1970s, series of works conducted by Friendenstein and coworkers on bone marrow aspirates demonstrated the relationship between osteogenic differentiation and a minor subpopulation of cells derived from bone marrow. 16 These cells were later proven to be distinguishable from the hematopoietic population and to be able to proliferate rapidly as adherent cells in tissue culture vessels. Another important breakthrough from Friendenstein’s team was the discovery that these cells could form the colony-forming unit when bone marrow was seeded as suspension culture following by differentiation into osteoblasts, adipocytes, and chondrocytes, suggesting that these cells confer the ability to proliferate and differentiate into different cell types. 17 In 1991, combined with the discovery of human embryonic stem cells (hESCs), which will be discussed in the next section, the term “mesenchymal stem cells”, previously known as stromal stem cells or “osteogenic” stem cells, was first coined in Caplan and widely used to date. 18 Starting with bone marrow transplantation 60 years ago, the journey of stem cell therapy has developed throughout the years to become a novel therapeutic agent of regenerative medicine to treat numerous incurable diseases, which will be reviewed and discussed in this review, including neurological disorders, pulmonary dysfunctions, metabolic/endocrine-related diseases, reproductive disorders, skin burns, and cardiovascular conditions).

Stem cell-based therapy: the history and cell source. a The timeline of major discoveries and advances in basic research and clinical applications of stem cell-based therapy. The term “stem cells” was first described in 1888, setting the first milestone in regenerative medicine. The hematopoietic progenitor cells were first discovered in 1902. In 1939, the first bone marrow transplantation was conducted in the treatment of aplasmic anemia. Since then, the translation of basic research to preclinical studies to clinical trials has driven the development of stem cell-based therapy by many discoveries and milestones. The isolations of “mesenchymal stem cells” in 1991 following by the discovery of human pluripotent stem cells have recently contributed to the progress of stem cell-based therapy in the treatment of human diseases. b Schematic of the different cell sources that can be used in stem cell-based therapy. (1) Human pluripotent stem cells, including embryonic stem cells (derived from inner cell mass of blastocyst) and induced pluripotent stem cells confer the ability to proliferate indefinitely in vitro and differentiate into numerous cell types of the human body, including three germ layers. (2) Mesenchymal stem cells are multipotent stem cells derived from mesoderm possessing self-renewal ability (limited in vitro) and differentiation potential into mesenchymal lineages. The differentiated/somatic cells can be reprogrammed back to the pluripotent stage using OSKM factors to generate induced pluripotent stem cells. It is important to note that stem cells show a relatively higher risk of tumor formation and lower risk of immune rejection (in the case of mesenchymal stem cells) when compared to that of somatic cells. The figure was created with BioRender.com

In this review, we described the different types of stem cell-based therapies (Fig. ​ (Fig.1b), 1b ), including hPSCs and MSCs, and provided an overview of their definition, history, and outstanding clinical applications. In addition, we further created the first literature portfolio for the “targeted therapy” of MSCs based on their origin, delineating their different tissue origins and downstream applications with an in-depth discussion of their mechanism of action. Finally, we provide our perspective on why the tissue origin of MSCs could contribute greatly to their downstream applications as a proposed hypothesis that needs to be proven or disproven in the future to further enhance the safety and effectiveness of stem cell-based therapy.

Stem cell-based therapy: an overview of current clinical applications

Cardiovascular diseases.

The clinical applications of stem cell-based therapies for heart diseases have been recently discussed comprehensively in the reviews 19 , 20 and therefore will be elaborated in this study as the focus discussions related to hPSCs and MSCs in the following sections. In general, the safety profiles of stem cell-based therapies are supported by a large body of preclinical and clinical studies, especially adult stem cell therapy (such as MSC-based products). However, clinical trials have not yet yielded data supporting the efficacy of the treatment, as numerous studies have shown paradoxical results and no statistically significant differences in infarct size, cardiac function, or clinical outcomes, even in phase III trials. 21 The results of a meta-analysis showed that stem cells derived from different sources did not exhibit any therapeutic effects on the improvement of myocardial contractility, cardiovascular remodeling, or clinical outcomes. 22 The disappointing results obtained from the clinical trials thus far could be explained by the fact that the administered cells may exert their therapeutic effects via an immune modulation rather than regenerative function. Thus, well-designed, randomized and placebo-controlled phase III trials with appropriate cell-preparation methods, patient selection, follow-up schedules and suitable clinical measurements need to be conducted to determine the efficacy of the treatments. In addition, concerns related to optimum cell source and dose, delivery route and timing of administration, cell distribution post administration and the mechanism of action also need to be addressed. In the following section of this review, we present clinical trials related to MSC-based therapy in cardiovascular disease with the aim of discussing the contradictory results of these trials and analyzing the potential challenges underlying the current approaches.

Digestive system diseases

Gastrointestinal diseases are among the most diagnosed conditions in the developed world, altering the life of one-third of individuals in Western countries. The gastrointestinal tract is protected from adverse substances in the gut environment by a single layer of epithelial cells that are known to have great regenerative ability in response to injuries and normal cell turnover. 23 These epithelial cells have a rapid turnover rate of every 2–7 days under normal conditions and even more rapidly following tissue damage and inflammation. This rapid proliferation ability is possible owing to the presence of a specific stem cell population that is strictly compartmentalized in the intestinal crypts. 24 The gastrointestinal tract is highly vulnerable to damage, tissue inflammation and diseases once the degradation of the mucosal lining layer occurs. The exposure of intestinal stem cells to the surrounding environment of the gut might result in the direct destruction of the stem cell layer or disruption of intestinal functions and lead to overt clinical symptoms. 25 In addition, the accumulation of stem cell defects as well as the presence of chronic inflammation and stress also contributes to the reduction of intestinal stem cell quality.

In terms of digestive disorders, Crohn’s disease (CD) and ulcerative colitis are the two major forms of inflammatory bowel disease (IBD) and represent a significant burden on the healthcare system. The former is a chronic, uncontrolled inflammatory condition of the intestinal mucosa characterized by segmental transmural mucosal inflammation and granulomatous changes. 26 The latter is a chronic inflammatory bowel disease affecting the colon and rectum, characterized by mucosal inflammation initiating in the rectum and extending proximal to the colon in a continuous fashion. 27 Cellular therapy in the treatment of CD can be divided into haematopoietic stem cell-based therapy and MSC-based therapy. The indication and recommendation of using HSCs for the treatment of IBD were proposed in 1995 by an international committee with four important criteria: (1) refractory to immunosuppressive treatment; (2) persistence of the disease conditions indicated via endoscopy, colonoscopy or magnetic resonance enterography; (3) patients who underwent an imminent surgical procedure with a high risk of short bowel syndromes or refractory colonic disease; and (4) patients who refused to treat persistent perianal lesions using coloproctectomy with a definitive stroma implant. 28 In the standard operation procedure, patents’ HSCs were recruited using cyclophosphamide, which is associated with granulocyte colony-stimulating factor (G-CSF), at two different administered concentrations (4 g/m 2 and 2 g/m 2 ). Recently, it was reported that high doses of cyclophosphamide do not improve the number of recruited HSCs but increase the risk of cardiac and bladder toxicity. An interest in using HSCTs in CD originated from case reports that autologous HSCTs can induce sustained disease remission in some 29 , 30 but not all patients 31 – 33 with CD. The first phase I trial was conducted in Chicago and recruited 12 patients with active moderate to severe CD refractory to conventional therapies. Eleven of 12 patients demonstrated sustained remission after a median follow-up of 18.5 months, and one patient developed recurrence of active CD. 31 The ASTIC trial (the Autologous Stem Cell Transplantation International Crohn Disease) was the first randomized clinical trial with the largest cohort of patients undergoing HSCT for refractory CD in 2015. 34 The early report of the trial showed no statistically significant improvement in clinical outcomes of mobilization and autologous HSCT compared with mobilization followed by conventional therapy. In addition, the procedure was associated with significant toxicity, leading to the suggestion that HSCT for patients with refractory CD should not be widespread. Interestingly, by using conventional assessments for clinical trials for CD, a group reassessed the outcomes of patients enrolled in the ASTIC trial showing clinical and endoscopic benefits, although a high number of adverse events were also detected. 35 A recent systematic review evaluated 18 human studies including 360 patients diagnosed with CD and showed that eleven studies confirmed the improvement of Crohn’s disease activity index between HSCT groups compared to the control group. 36 Towards the cell sources, HSCs are the better sources as they afforded more stable outcomes when compared to that of MSC-based therapy. 37 Moreover, autologous stem cells were better than their allogeneic counterparts. 36 The safety of stem cell-based therapy in the treatment of CD has attracted our attention, as the risk of infection in patients with CD was relatively higher than that in those undergoing administration to treat cancer or other diseases. During the stem cell mobilization process, patient immunity is significantly compromised, leading to a high risk of infection, and requires carefully nursed and suitable antibiotic treatment to reduce the development of adverse events. Taken together, stem cell-based therapy for digestive disease reduced inflammation and improved the patient’s quality of life as well as bowel functions, although the high risk of adverse events needs to be carefully monitored to further improve patient safety and treatment outcomes.

Liver diseases

The liver is the largest vital organ in the human body and performs essential biological functions, including detoxification of the organism, metabolism, supporting digestion, vitamin storage, and other functions. 38 The disruption of liver homeostasis and function might lead to the development of pathological conditions such as liver failure, cirrhosis, cancer, alcoholic liver disease, nonalcoholic fatty liver disease (NAFLD), and autoimmune liver disease (ALD). Orthotropic liver transplantation is the only effective treatment for severe liver diseases, but the number of available and suitable donor organs is very limited. Currently, stem cell-based therapies in the treatment of liver disease are associated with HSCs, MSCs, hPSCs, and liver progenitor cells.

Liver failure is a critical condition characterized by severe liver dysfunctions or decompensation caused by numerous factors with a relatively high mortality rate. Stem cell-based therapy is a novel alternative approach in the treatment of liver failure, as it is believed to participate in the enhancement of liver regeneration and recovery. The results of a meta-analysis including four randomized controlled trials and six nonrandomized controlled trials in the treatment of acute-on-chronic liver failure (ACLF) demonstrated that clinical outcomes of stem cell therapy were achieved in the short term, requiring multiple doses of stem cells to prolong the therapeutic effects. 39 , 40 Interestingly, although MSC-based therapies improved liver functions, including the model of end-stage liver disease score, albumin level, total bilirubin, and coagulation, beneficial effects on survival rate and aminotransferase level were not observed. 41 A randomized controlled trial illustrated the improvement of liver functions and reduction of severe infections in patients with hepatitis B virus-related ACLF receiving allogeneic bone marrow-derived MSCs (BM-MSCs) via peripheral infusion. 42 HSCs from peripheral blood after the G-CSF mobilization process were used in a phase I clinical trial and exhibited an improvement in serum bilirubin and albumin in patients with chronic liver failure without any specific adverse events related to the administration. 43 Taken together, an overview of stem cell-based therapy in the treatment of liver failure indicates the potential therapeutic effects on liver functions with a strong safety profile, although larger randomized controlled trials are still needed to assure the conclusions.

Liver cirrhosis is one of the major causes of morbidity and mortality worldwide and is characterized by diffuse nodular regeneration with dense fibrotic septa and subsequent parenchymal extinction leading to the collapse of liver vascular structure. 44 In fact, liver cirrhosis is considered the end-stage of liver disease that eventually leads to death unless liver transplantation is performed. Stem cell-based therapy, especially MSCs, currently emerges as a potential treatment with encouraging results for treating liver cirrhosis. In a clinical trial using umbilical cord-derived MSCs (UC-MSCs), 45 chronic hepatitis B patients with decompensated liver cirrhosis were divided into two groups: the MSC group ( n  = 30) and the control group ( n  = 15). 45 The results showed a significant reduction in ascites volume in the MSC group compared with the control. Liver function was also significantly improved in the MSC groups, as indicated by the increase in serum albumin concentration, reduction in total serum bilirubin levels, and decrease in the sodium model for end-stage liver disease score. 45 Similar results were also reported from a phase II trial using BM-MSCs in 25 patients with HCV-induced liver cirrhosis. 46 Consistent with these studies, three other clinical trials targeting liver cirrhosis caused by hepatitis B and alcoholic cirrhosis were conducted and confirmed that MSC administration enhanced and recovered liver functions. 47 – 49 With the large cohort study as the clinical trial conducted by Fang, the safety and potential therapeutic effects of MSC-based therapies could be further strengthened and confirmed the feasibility of the treatment in virus-related liver cirrhosis. 49 In terms of delivery route, a randomized controlled phase 2 trial suggested that systemic delivery of BM-MSCs does not show therapeutic effects on patients with liver cirrhosis. 50 MSCs are not the only cell source for liver cirrhosis. Recently, an open-label clinical trial conducted in 19 children with liver cirrhosis due to biliary atresia after the Kasai operation illustrated the safety and feasibility of the approach by showing the improvement of liver function after bone marrow mononuclear cell (BMNC) administration assessed by biochemical tests and pediatric end-stage liver disease (PELD) scores. 51 Another study using BMNCs in 32 decompensated liver cirrhosis patients illustrated the safety and effectiveness of BMNC administration in comparison with the control group. 52 Recently, a long-term analysis of patients receiving peripheral blood-derived stem cells indicated a significant improvement in the long-term survival rate when compared to the control group, and the risk of hepatocellular carcinoma formation did not increase. 53 CD133 + HSC infusion was performed in a multicentre, open, randomized controlled phase 2 trial in patients with liver cirrhosis; the results did not support the improvement of liver conditions, and cirrhosis persisted. 54 Notably, these results are in line with a previous randomized controlled study, which also reported that G-CSF and bone marrow-derived stem cells delivered via the hepatic artery did not introduce therapeutic potential as expected. 55 Thus, stem cell-based therapy for liver cirrhosis is still in its immature stage and requires larger trials with well-designed experiments to confirm the efficacy of the treatment.

Nonalcoholic fatty liver disease (NAFLD) is the most common medical condition caused by genetic and lifestyle factors and results in a severe liver condition and increased cardiovascular risk. 56 NAFLD is the hidden enemy, as most patients are asymptomatic for a long time, and their routine life is unaffected. Thus, the detection, identification, and management of NAFLD conditions are challenging tasks, as patients diagnosed with NAFLD often develop nonalcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma. 57 Although preclinical studies have shown that stem cell administration could enhance liver function in NAFLD models, a limited number of clinical trials were performed in human subjects. Recently, a multi-institutional clinical trial using freshly isolated autologous adipose tissue-derived regenerative cells was performed in Japan to treat seven NAFLD patients. 58 The results illustrated the improvement in the serum albumin level of six patients and prothrombin activity of five patients, and no treatment-related adverse events or severe adverse events were observed. This study illustrates the therapeutic potential of stem cell-based therapy in the treatment of NAFLD.

Autoimmune liver disease (ALD) is a severe liver condition affecting children and adults worldwide, with a female predominance. 59 The condition occurs in genetically predisposed patients when a stimulator, such as virus infection, leads to a T-cell-mediated autoimmune response directed against liver autoantigens. As a result, patients with ALD might develop liver cirrhosis, hepatocellular carcinoma, and, in severe cases, death. To date, HSCT and bone marrow transplantation are the two common stem cell-based therapies exhibiting therapeutic potential for ALD in clinical trials. An interesting report illustrated that haploidentical HSCTs could cure ALD in patients with sickle cells. 60 This report is particularly important, as it illustrates the potential therapeutic approach of using haploidentical HSCTs to treat patients with both sickle cells and ALD. Another case report described a 19-year-old man with a 4-year history of ALD who developed acute lymphoblastic leukemia and required allogeneic bone marrow transplantation from this wholesome brother. 61 The clinical data showed that immunosuppressive therapy for transplantation generated ALD remission in the patient. 62 However, the data also provided valid information related to the sustained remission and the normalization of ASGPR-specific suppressor-inducer T-cell activity following bone marrow transplantation, suggesting that these suppressor functions originated from donor T cells. 61 Thus, it was suggested that if standard immunosuppressive treatment fails, alternative cellular immunotherapy would be a viable option for patients with ALD. Primary biliary cholangitis (PBC), usually known as primary biliary cirrhosis, is a type of ALD characterized by a slow, progressive destruction of small bile ducts of the liver leading to the formation of cirrhosis and accumulation of bile and other toxins in the liver. A pilot, single-arm trial from China recruited seven patents with PBC who had a suboptimal response to ursodeoxycholic acid (UDCA) treatment. 63 These patients received UDCA treatment in combination with three rounds of allogeneic UC-MSCs at 4-week intervals with a dose of 0.5 × 10 6 cells/kg of patient body weight via the peripheral vein. No treatment-related adverse events or severe adverse events were observed throughout the course of the study. The clinical data indicated significant improvement in liver function, including reduction of serum ALP and gamma-glutamyltransferase (GGT) at 48 weeks post administration. The common symptoms of PBC, including fatigue, pruritus, and hypogastric ascites volume, were also reduced, supporting the feasibility of MSC-based therapy in the treatment of PBC, although major limitations of the study were nonrandomized, no control group and small sample size. Another study was conducted in China with ten PBC patients who underwent incompetent UDCA treatment for more than 1 year. These patients received a range of 3–5 allogeneic BM-MSCs/kg body weight by intravenous infusion. 64 Although these early studies have several limitations, such as small sample size, nonrandomization, and no control group, their preliminary data related to safety and efficacy herald the prospects and support the feasibility of stem cell-based therapy in the treatment of ALD.

In summary, the current number of trials for liver disease using stem cell-based therapy has provided fundamental data supporting the safety and potential therapeutic effects in various liver diseases. Unfortunately, due to the small number of trials, several obstacles need to be overcome to prove the effectiveness of the treatments, including (1) stem cell source and dose, (2) administration route, (3) time of intervention, and (4) clinical assessments during the follow-up period. Only by addressing these challenges we will be able to prove, facilitate and promote stem cell-based therapy as a mainstream treatment for liver diseases.

Arthritis is a general term describing cartilage conditions that cause pain and inflammation of the joints. Osteoarthritis (OA) is the most common form of arthritis caused by persistent degeneration and poor recovery of articular cartilage. 65 OA affects one or several diarthrodial joints, such as small joints at the hand and large joints at the knee and hips, leading to severe pain and subsequent reduction in the mobility of patients. There are two types of OA: (1) primary OA or idiopathic OA and secondary OA caused by causative factors such as trauma, surgery, and abnormal joint development at birth. 66 As conventional treatments for OA are not consistent in their effectiveness and might cause unbearable pain as well as long-term rehabilitation (in the case of joint replacement), there is a need for a more reliable, less painful, and curative therapy targeting the root of OA. 67 Thus, stem cell therapy has recently emerged as an alternative approach for OA and has drawn great attention in the regenerative field.

The administration of HSCs has been proven to reduce bone lesions, enhance bone regeneration and stimulate the vascularization process in degenerative cartilage. Attempts were made to evaluate the efficacy of peripheral blood stem cells in ten OA patients by three intraarticular injections. Post-administration analysis indicated a reduction in the WOMAC index with a significant reduction in all parameters. All patients completed 6-min walk tests with an increase of more than 54 meters. MRI analysis indicated an improvement in cartilage thickness, suggesting that cartilage degeneration was reduced post administration. To further enhance the therapeutic potential of HSCT, CD34 + stem cells were proposed to be used in combination with the rehabilitation algorithm, which included three stages: preoperative, hospitalization and outpatient periods. 68 Currently, a large wave of studies has been directed to MSC-based therapy for the treatment of OA due to their immunoregulatory functions and anti-inflammatory characteristics. MSCs have been used as the main cell source in several multiple and small-scale trials, proving their safety profile and potential effectiveness in alleviating pain, reducing cartilage degeneration, and enhancing the regeneration of cartilage structure and morphology in some cases. However, the best source of MSCs, whether from bone marrow, adipose tissue, or umbilical cord, for the management of OA is still a great question to be answered. A systematic review investigating over sixty-one of 3172 articles with approximately 2390 OA patients supported the positive effects of MSC-based therapy on OA patients, although the study also pointed out the fact that these therapeutic potentials were based on limited high-quality evidence and long-term follow-up. 69 Moreover, the study found no obvious evidence supporting the most effective source of MSCs for treating OA. Another systematic review covering 36 clinical trials, of which 14 studies were randomized trials, provides an interesting view in terms of the efficacy of autologous MSC-based therapy in the treatment of OA. 70 In terms of BM-MSCs, 14 clinical trials reported the clinical outcomes at the 1-year follow-up, in which 57% of trials reported clinical outcomes that were significantly better in comparison with the control group. However, strength analysis of the data set showed that outcomes from six trials were low, whereas the outcomes of the remaining eight trials were extremely low. Moreover, the positive evidence obtained from MRI analysis was low to very low strength of evidence after 1-year post administration. 70 Similar results were also found in the outcome analysis of autologous adipose tissue-derived MSCs (AT-MSCs). Thus, the review indicated low quality of evidence for the therapeutic potential of MSC therapy on clinical outcomes and MRI analysis. The low quality of clinical outcomes could be explained by the differences in interventions (including cell sources, cell doses, and administration routes), combination treatments (with hyaluronic acid, 71 peripheral blood plasma, 72 etc.), control treatments and clinical outcome measurements between randomized clinical trials. 73 In addition, the data of the systematic analysis could not prove the better source of MSCs for OA treatment. Taken together, although stem cell-based therapy has been shown to be safe and feasible in the management of OA, the authors support the notion that stem cell-based therapy could be considered an alternative treatment for OA when first-line treatments, such as education, exercise, and body weight management, have failed.

Cancer treatment

Stem cell therapy in the treatment of cancer is a sensitive term and needs to be used and discussed with caution. Clinicians and researchers should protect patients with cancer from expensive and potentially dangerous or ineffective stem cell-based therapy and patients without a cancer diagnosis from the risk of malignancy development. In general, unproven stem cell clinics employed three cell-based therapies for cancer management, including autologous HSCTs, stromal vascular fraction (SVF), and multipotent stem cells, such as MSCs. Allogeneic HSCTs confer the ability to generate donor lymphocytes that contribute to the suppression and regression of hematological malignancies and select solid tumors, a specific condition known as “graft-versus-tumor effects”. 74 However, stem cell clinics provide allogeneic cell-based therapy for the treatment of solid malignancies despite limited scientific evidence supporting the safety and efficacy of the treatment. High-quality evidence from the Cochrane library shows that marrow transplantation via autologous HSCTs in combination with high-dose chemotherapy does not improve the overall survival of women with metastatic breast cancer. In addition, a study including more than 41,000 breast cancer patients demonstrated no significant difference in survival benefits between patients who received HSCTs following high-dose chemotherapy and patients who underwent conventional treatment. 75 Thus, the use of autologous T-cell transplants as monotherapy and advertising stem cell-based therapies as if they are medically approved or preferred treatment of solid tumors is considered untrue statements and needs to be alerted to cancer patients. 76

Over the past decades, many preclinical studies have demonstrated the potential of MSC-based therapy in cancer treatment due to their unique properties. They confer the ability to migrate toward damaged sites via inherent tropism controlled by growth factors, chemokines, and cytokines. MSCs express specific C–X–C chemokine receptor type 4 (CXCR4) and other chemokine receptors (including CCR1, CCR2, CCR4, CCR7, etc.) that are essential to respond to the surrounding signals. 77 In addition, specific adherent proteins, including CD49d, CD44, CD54, CD102, and CD106, are also expressed on the MSC surface, allowing them to attach, rotate, migrate, and penetrate the blood vessel lumen to infiltrate the damaged tissue. 78 Similar to damaged tissues, tumors secrete a wide range of chemoattractant that also attract MSC migration via the CXCL12/CXCR4 axis. Previous studies also found that MSC migration toward the cancer site is tightly controlled by diffusible cytokines such as interleukin 8 (IL-8) and growth factors including transforming growth factor-beta 1 (TGF-β1), 79 platelet-derived growth factor (PDGF), 80 fibroblast growth factor 2 (FGF-2), 81 vascular endothelial growth factor (VEGF), 81 and extracellular matrix molecules such as matrix metalloproteinase-2 (MMP-2). 82 Once MSCs migrate successfully to cancerous tissue, accumulating evidence demonstrates the interaction between MSCs and cancer cells to exhibit their protumour and antitumour effects, which are the major concerns of MSC-based therapy. MSCs are well-known for their regenerative effects that regulate tissue repair and recovery. This unique ability is also attributed to the protumour functions of these cells. A previous study reported that breast cancer cells induce MSC secretion of chemokine (C–C motif) ligand 5 (CCL-5), which regulates the tumor invasion process. 83 , 84 Other studies also found that MSCs secrete a wide range of growth factors (VEGF, basic FGF, HGF, PDGF, etc.) that inhibits apoptosis of cancer cells. 85 Moreover, MSCs also respond to signals released from cancer cells, such as TGF-β, 86 to transform into cancer-associated fibroblasts, a specific cell type residing within the tumor microenvironment capable of promoting tumorigenesis. 87 Although MSCs have been proven to be involved in protumour activities, they also have potent tumor suppression abilities that have been used to develop cancer treatments. It has been suggested that MSCs exhibit their tumor inhibitory effects by inhibiting the Wnt and AKT signaling pathways, 88 reducing the angiogenesis process, 89 stimulating inflammatory cell infiltration, 90 and inducing tumor cell cycle arrest and apoptosis. 91 To date, the exact functions of MSCs in both protumour and antitumor activities are still a controversial issue across the stem cell field. Other approaches exploit gene editing and tissue engineering to convert MSCs into “a Trojan horse” that could exhibit antitumor functions. In addition, MSCs can also be modified to express specific anticancer miRNAs exhibiting tumor-suppressive behaviors. 92 However, genetically modified MSCs are still underdeveloped and require intensive investigation in the clinical setting.

To date, ~25 clinical trials have been registered on ClinicalTrials.gov aimed at using MSCs as a therapeutic treatment for cancer. 93 These trials are mostly phase 1 and 2 studies focusing on evaluating the safety and efficacy of the treatment. Studies exploiting MSC-based therapy have combined MSCs with an oncolytic virus approach. Oncolytic viruses are specific types of viruses that can be genetically engineered or naturally present, conferring the ability to selectively infect cancer cells and kill them without damaging the surrounding healthy cells. 94 A completed phase I/II study using BM-MSCs infected with the oncolytic adenovirus ICOVIR5 in the treatment of metastatic and refractory solid tumors in children and adult patients demonstrated the safety of the treatment and provided preliminary data supporting their therapeutic potential. 95 The same group also reported a complete disappearance of all signs of cancer in response to MSC-based therapy in one pediatric case three years post administration. 96 A reported study in 2019 claimed that adipose-derived MSCs infected with vaccinia virus have the potential to eradicate resistant tumor cells via the combination of potent virus amplification and senitization of the tumor cells to virus infection. 97 However, in a recently published review, a valid question was posed regarding the 2019 study that “do these reported data merit inclusion in the publication record when they were collected by such groups using a dubious therapeutic that was eventually confiscated by US Marshals?” 76

Taken together, cancer research and therapy have entered an innovative and fascinating era with advancements in traditional therapies such as chemotherapy, radiotherapy, and surgery on one hand and stem cell-based therapy on the other hand. Although stem cell-based therapy has been considered a novel and attractive therapeutic approach for cancer treatment, it has been hampered by contradictory results describing the protumour and antitumour effects in preclinical studies. Despite this contradictory reality, the use of stem cell-based therapy, especially MSCs, offers new hope to cancer patients by providing a new and more effective tool in personalized medicine. The authors support the use of MSC-based therapy as a Trojan horse to deliver specific anticancer functions toward cancer cells to suppress their proliferation, eradicate cancer cells, or limit the vascularization process of cancerous tissue to improve the clinical safety and efficacy of the treatment.

Human pluripotent stem cell-based therapy: a growing giant

The discovery of hPSCs, including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), has revolutionized stem cell research and cell-based therapy. 98 hESCs were first isolated from blastocyst-stage embryos in 1998, 99 followed by breakthrough reprogramming research that converted somatic cells into hiPSCs using just four genetic factors. 100 , 101 Methods have been developed to maintain these cells long-term in vitro and initiate their differentiation into a wide variety of cell types, opening a new era in regenerative medicine, particularly cell therapy to replace lost or damaged tissues.

History of hPSCs

hPSCs are defined as self-renewable cell types that confer the ability to differentiate into various cellular phenotypes of the human body, including three germ layers. 102 Historically, the first pluripotent cell lines to be generated were embryonic carcinoma (EC) cell lines established from human germ cell tumors 103 and murine undifferentiated compartments. 104 Although EC cells are a powerful tool in vitro, these cells are not suitable for clinical applications due to their cancer-derived origin and aneuploidy genotype. 105 The first murine ESCs were established in 1981 based on the culture techniques obtained from EC research. 106 Murine ESCs are derived from the inner cell mass (ICM) of the pre-implantation blastocyst, a unique biological structure that contains outer trophoblast layers that give rise to the placenta and ICM. 107 In vivo ESCs only exist for a short period during the embryo’s development, and they can be isolated and maintained indefinitely in vitro in an undifferentiated state. The discovery of murine ESCs has dramatically changed the field of biomedical research and regenerative medicine over the last 40 years. Since then, enormous investigations have been made to isolate and culture ESCs from other species, including hESCs, in 1998. 99 The success of Thomson et al. in 1998 triggered the great controversy in media and ethical research boards across the globe, with particularly strong objections being raised to the use of human embryos for research purposes. 108 Several studies using hESCs have been conducted demonstrating their therapeutic potential in the clinical setting. However, the use of hESCs is limited due to (1) the ethical barrier related to the destruction of human embryos and (2) the potential risk of immunological rejection, as hESCs are isolated from pre-implantation blastocysts, which are not autologous in origin. To overcome these two great obstacles, several research groups have been trying to develop technology to generate hESCs, including nuclear transfer technology, the well-known strategy that creates Dolly sheep, although the generation of human nuclear transfer ESCs remains technically challenging. 109 Taking a different approach, in 2006, Yamanaka and Takahashi generated artificial PSCs from adult and embryonic mouse somatic cells using four transcription factors ( Oct-3/4 , Sox2 , Klf4 , and c-Myc , called OSKM) reduced from 24 factors. 100 Thereafter, in 2007, Takahashi and colleagues successfully generated the first hiPSCs exhibiting molecular and biological features similar to those of hESCs using the same OSKM factors. 101 Since then, hiPSCs have been widely studied to expand our knowledge of the pathogenesis of numerous diseases and aid in developing new cell-based therapies as well as personalized medicine.

Clinical applications of hPSCs

Since its beginning 24 years ago, hPSC research has evolved momentously toward applications in regenerative medicine, disease modeling, drug screening and discovery, and stem cell-based therapy. In clinical trial settings, the uses of hESCs are restricted by ethical concerns and tight regulation, and the limited preclinical data support their therapeutic potential. However, it is important to acknowledge several successful outcomes of hESC-based therapies in treating human diseases. In 2012, Steven Schwartz and his team reported the first clinical evidence of using hESC-derived retinal pigment epithelium (RPE) in the treatment of Stargardt’s macular dystrophy, the most common pediatric macular degeneration, and an individual with dry age-related macular degeneration. 110 , 111 With a differentiation efficiency of RPE greater than 99%, 5 × 10 4 RPEs were injected into the subretinal space of one eye in each patient. As the hESC source of RPE differentiation was exposed to mouse embryonic stem cells, it was considered a xenotransplantation product and required a lower dose of immunosuppression treatment. This study showed that hESCs improved the vision of patients by differentiating into functional RPE without any severe adverse events. The trial was then expanded into two open-label, phase I/II studies with the published results in 2015 supporting the primary findings. 112 In these trials, patients were divided into three groups receiving three different doses of hESC-derived RPE, including 10 × 10 4 , 15 × 10 4 and 50 × 10 4 RPE cells per eye. After 22 months of follow-up, 19 patients showed improvement in eyesight, seven patients exhibited no improvement, and one patient experienced a further loss of eyesight. The technical challenge of hESC-derived RPE engraftment was an unbalanced proliferation of RPE post administration, which was observed in 72% of treated patients. A similar approach was also conducted in two South Korean patients diagnosed with age-induced macular degeneration and two patients with Stargardt macular dystrophy. 113 The results supported the safety of hESC-derived RPE cells and illustrated an improvement in visual acuity in three patients. Recently, clinical-graded hESC-derived RPE cells were also developed by Chinese researchers under xeno-free culture conditions to treat patients with wet age-related degeneration. 114 As hESC development is still associated with ethical concerns and immunological complications related to allogeneic administration, hiPSC-derived RPE cells have emerged as a potential cell source for macular degeneration. Although RPE differentiation protocols have been developed and optimized to improve the efficacy of hiPSC-derived RPE cells, they are still insufficient, time-consuming and labor intensive. 115 , 116 For clinical application, an efficient differentiation of “primed” to “naïve” state hiPSCs toward the RPE was developed using feeder-free culture conditions utilizing the transient inhibition of the FGF/MAPK signaling pathway. 117 Overexpression of specific transcription factors in hiPSCs throughout the differentiation process is also an interesting approach to generate a large number of RPE cells for clinical use. In a recent study, overexpression of three eye-field transcription factors, including OTX2, PAX6, and MITF , stimulated RPE differentiation in hiPSCs and generated functional RPE cells suitable for transplantation. 118 To date, although reported data from phase I/II clinical trials have been produced enough to support the safety of hESC-derived RPE cells, the treatment is still in its immature stage. Thus, future studies should focus on the development of the cellular manufacturing process of RPE and the subretinal administration route to further improve the outcomes of RPE fabrication and engraftment into the patient’s retina (recommended review 119 ).

Numerous studies have demonstrated that hESC-derived cardiomyocytes exhibit cardiac transcription factors and display a cardiomyocyte phenotype and immature electrical phenotype. In addition, using hPSC-derived cardiomyocytes could provide a large number of cells required for true remuscularization and transplantation. Thus, these cells can be a promising novel therapeutic approach for the treatment of human cardiovascular diseases. In a case report, hESC-derived cardiomyocytes showed potential therapeutic effects in patients with severe heart failure without any subsequent complications. 120 This study was a phase I trial (ESCORT [Transplantation of Human Embryonic Stem Cell-derived Progenitors in Severe Heart Failure] trial) to evaluate the safety of cardiomyocyte progenitor cells derived from hESCs seeded in fibrin gel scaffolds for 10 patients with severe heart failure ( {"type":"clinical-trial","attrs":{"text":"NCT02057900","term_id":"NCT02057900"}} NCT02057900 ). The encouraging results from this study demonstrated the feasibility of producing hESC-derived cardiomyocyte progenitor cells toward clinical-grade standards and combining them with a tissue-engineered scaffold to treat severe heart disease (the first patient of this trial has already reached the 7-year follow-up in October 2021). 121 Currently, the two ongoing clinical trials using hPSC-derived cardiomyocytes have drawn great attention, as their results would pave the way to lift the bar for approving therapies for commercial use. The first trial was conducted by a team led by cardiac surgeon Yoshiki Sawa at Osaka University using hiPSC-derived cardiomyocytes embedded in a cell sheet for engraftment (jRCT2052190081). The trials started first with three patients followed by ten patients to assess the safety of the approach. Once safety is met, the treatment can be sold commercially under Japan’s fast-track system for regenerative medicine. 122 Another trial used a collagen-based construct called BioVAT-HF to contain hiPSC-derived cardiomyocytes. The trial was divided into two parts to evaluate the cell dose: (Part A) recruiting 18 patients and (Part B) recruiting 35 patients to test a broad range of engineered human myocardium (EHM) doses. The expected results from this study will provide the “proof-of-concept” for the use of EHM in the stimulation of heart remuscularization in humans. To date, no adverse events or severe adverse events have been reported from these trials, supporting the safety of the procedure. However, as the number of treated patients was relatively small, limitations in drawing conclusions regarding efficacy are not yet possible. 21 , 123

One of the first clinical trials using hPSC-based therapy was conducted by Geron Corporation in 2010 using hESC-derived oligodendrocyte progenitor cells (OPC1) to treat spinal cord injury (SCI). The results confirmed the safety one year post administration in five participants, and magnetic resonance imaging demonstrated improvement of spinal cord deterioration in four participants. 124 Asterias Biotherapeutic (AST) continued the Geron study by conducting the SCiStar Phase I/IIa study to evaluate the therapeutic effects of AST-OPC1 ( {"type":"clinical-trial","attrs":{"text":"NCT02302157","term_id":"NCT02302157"}} NCT02302157 ). The trial’s results published in clinicaltrials.gov demonstrated significant improvement in running speed, forelimb stride length, forelimb longitudinal deviations, and rear stride frequency. Interestingly, the recently published data of a phase 1, multicentre, nonrandomized, single-group assignment, interventional trial illustrated no evidence of neurological decline, enlarging masses, further spinal cord damage, or syrinx formation in patients 10 years post administration of the OPC1 product. 125 This data set provides solid evidence supporting the safety of OPC1 with an event-free period of up to 10 years, which strengthens the safety profile of the SCiStar trial.

Analysis of the global trends in clinical trials using hPSC-based therapy showed that 77.1% of studies were observational (no cells were administered into patient), and only 22.9% of studies used hPSC-derived cells as interventional treatment. 126 The number of studies using hiPSCs was relatively higher than that using hESCs, which was 74.8% compared to 25.2%, respectively. The majority of observational studies were performed in developed countries, including the USA (41.6%) and France (16.8%), whereas interventional studies were conducted in Asian countries, including China (36.7%), Japan (13.3%), and South Korea (10%). The trends in therapeutic studies were also clear in terms of targeted diseases. The three most studied diseases were ophthalmological conditions, circulatory disorders, and nervous systems. 127 However, it is surprising that the clinical applications of hPSCs have achieved little progress since the first hESCs were discovered worldwide. The relatively low number of clinical trials focusing on using iPSCs as therapeutic agents to administer into patients could be ascribed to the unstable genome of hiPSCs, 128 immunological rejection, 129 and the potential for tumor formation. 130

Mesenchymal stem/stromal cell-based therapy: is it time to consider their origin toward targeted therapy?

Approximately 55 years ago, fibroblast-like, plastic-adherent cells, later named mesenchymal stem cells (MSCs) by Arnold L. Caplan, 18 were discovered for the first time in mouse bone marrow (BM) and were later demonstrated to be able to form colony-like structures, proliferate, and differentiate into bone/reticular tissue, cartilage, and fat. 131 Protocols were subsequently established to directly culture this subpopulation of stromal cells from BM in vitro and to stimulate their differentiation into adipocytes, chondroblasts, and osteoblasts. 132 Since then, MSCs have been found in and derived from different human tissue sources, including adipose tissue (AT), the umbilical cord (UC), UC blood, the placenta, dental pulp, amniotic fluid, etc. 133 To standardize and define MSCs, the International Society for Cell and Gene Therapy (ISCT) set minimal identification criteria for MSCs derived from multiple tissue sources. 134 Among them, MSCs derived from AT, BM, and UC are the most commonly studied MSCs in human clinical trials, 135 and they constitute the three major tissue sources of MSCs that will be discussed in this review.

The discovery of MSCs opened an era during which preclinical studies and clinical trials have been performed to assess the safety and efficacy of MSCs in the treatment of various diseases. The major conclusion of these studies and trials is that MSC-based therapy is safe, although the outcomes have usually been either neutral or at best marginally positive in terms of the clinically relevant endpoints regardless of MSC tissue origin, route of infusion, dose, administration duration, and preconditioning. 136 It is important to note that a solid background of knowledge has been generated from all these studies that has fueled the recent translational research in MSC-based therapy. As MSCs have been intensively studied over the last 55 years and have become the subject of multiple reviews, systematic reviews, and meta-analyses, the objective of this paper is not to duplicate these publications. Rather, we will discuss the questions that both clinicians and researchers are currently exploring with regard to MSC-based therapy, diligently seeking answers to the following:

• “With a solid body of data supporting their safety profiles derived from both preclinical and clinical studies, does the tissue origin of MSCs also play a role in their downstream clinical applications in the treatment of different human diseases?”
• “Do MSCs derived from AT, BM, and UC exhibit similar efficacy in the treatment of neurological diseases, metabolic/endocrine-related disorders, reproductive dysfunction, skin burns, lung fibrosis, pulmonary disease, and cardiovascular conditions?”

To answer these questions, we will first focus on the most recently published clinical data regarding these targeted conditions, including neurological disorders, pulmonary dysfunctions, metabolic/endocrine-related diseases, reproductive disorders, skin burns, and heart-related diseases, to analyze the potential efficacy of MSCs derived from AT, BM, and UC. Based on the level of clinical improvement observed in each trial, we analyzed the potential efficacy of MSCs derived from each source to visualize the correlation between patient improvement and MSC sources. We will then address recent trends in the exclusive use of MSC-based products, focusing on the efficacy of treatment with MSCs from each of the abovementioned sources, and we will analyze the relationship between the respective efficacies of MSCs from these sources in relation to the targeted disease conditions. Finally, we propose a hypothesis and mechanism to achieve the currently still unmet objective of evaluating the use of MSCs from AT, BM, and UC in regenerative medicine.

An overview of MSC tissue origins and therapeutic potential

In general, MSCs are reported to be isolated from numerous tissue types, but all of these types can be organized into two major sources: adult 137 and perinatal sources 138 (Fig. ​ (Fig.2). 2 ). Adult sources of MSCs are defined as tissues that can be harvested or obtained from an individual, such as dental pulp, 139 BM, peripheral blood, 140 AT, 141 lungs, 142 hair, 143 or the heart. 144 Adult MSCs usually reside in specialized structures called stem cell niches, which provide the microenvironment, growth factors, cell-to-cell contacts and external signals necessary for maintaining stemness and differentiation ability. 145 BM was the first adult source of MSCs discovered by Friedenstein 131 and has become one of the most documented and largely used MSC sources to date, followed by AT. BM-MSCs are isolated and cultured in vitro from BM aspirates using a Ficoll gradient-centrifugation method 146 or a red blood cell lysate buffer to collect BM mononuclear cell populations, whereas AT-MSCs are obtained from stromal vascular fractions of enzymatically digested AT obtained through liposuction, 141 lipoplasty, or lipectomy procedures. 147 These tissue collection procedures are invasive and painful for the patient and are accompanied by a risk of infection, although BM aspiration and adipose liposuction are considered safe procedures for BM and AT biopsies. The number of MSCs that can be isolated from these adult tissues varies significantly in a tissue-dependent manner. The percentage of MSCs in BM mononuclear cells ranges from 0.001 to 0.01% following gradient centrifugation. 132 The number of MSCs in AT is at least 500 times higher than that in BM, with approximately 5,000 MSCs per 1 g of AT. Perinatal sources of MSCs consist of UC-derived components, such as UC, Wharton’s jelly, and UC blood, and placental structures, such as the placental membrane, amnion, chorion membrane, and amniotic fluid. 138 The collection of perinatal MSCs, such as UC-MSCs, is noninvasive, as the placenta, UC, UC blood, and amnion are considered waste products that are usually discarded after birth (with no ethical barriers). 148 Although MSCs represent only 10 −7 % the cells found in UC, their higher proliferation rate and rapid population doubling time allow these cells to rapidly replicate and increase in number during in vitro culture. 149 Under standardized xeno-free and serum-free culture platforms, AT-MSCs show a faster proliferation rate and a higher number of colony-forming units than BM-MSCs. 149 UC-MSCs have the fastest population doubling time compared to AT-MSCs and BM-MSCs in both conventional culture conditions and xeno- and serum-free environments. 149 MSCs extracted from AT, BM and UC exhibit all minimal criteria listed by the ISCT, including morphology (plastic adherence and spindle shape), MSC surface markers (95% positive for CD73, CD90 and CD105; less than 2% negative for CD11, CD13, CD19, CD34, CD45, and HLR-DR) and differentiation ability into chondrocytes, osteocytes, and adipocytes. 150

The two major sources of MSCs: adult and perinatal sources. The adult sources of MSCs are specific tissue in human body where MSCs could be isolated, including bone marrow, adipose tissue, dental pulp, peripheral blood, menstrual blood, muscle, etc. The perinatal sources of MSCs consist of umbilical cord-derived components, such as umbilical cord, Wharton’s jelly, umbilical cord blood, and placental structures, such as placental membrane, amnion, chorion membrane, amniotic fluid, etc. The figure was created with BioRender.com

In fact, although MSCs derived from either adult or perinatal sources exhibit similar morphology and the basic characteristics of MSCs, studies have demonstrated that these cells also differ from each other. Regarding immunophenotyping, AT-MSCs express high levels of CD49d and low levels of Stro-1. An analysis of the expression of CD49d and CD106 showed that the former is strongly expressed in AT-MSCs, in contrast to BM-MSCs, whereas CD106 is expressed in BM-MSCs but not in AT-MSCs. 151 Increased expression of CD133, which is associated with stem cell regeneration, differentiation, and metabolic functions, 152 was observed in BM-MSCs compared to MSCs from other sources. 153 A recent study showed that CD146 expression in UC-MSCs was higher than that in AT- and BM-MSCs, 153 supporting the observation that UC-MSCs have a stronger attachment and a higher proliferation rate than MSCs from other sources, as CD146 is a key cell adhesion protein in vascular and endothelial cell types. 154 In terms of differentiation ability, donor-matched BM-MSCs exhibit a higher ability to differentiate into chondrogenic and osteogenic cell types than AT-MSCs, whereas AT-MSCs show a stronger capacity toward the adipogenic lineage. 150 The findings from an in vitro differentiation study indicated that BM-MSCs are prone to osteogenic differentiation, whereas AT-MSCs possess stronger adipogenic differentiation ability, which can be explained by the fact that the epigenetic memory obtained from either BM or AT drives the favored MSC differentiation along an osteoblastic or adipocytic lineage. 155 Interestingly, although UC-MSCs have the ability to differentiate into adipocytes, osteocytes, or chondrocytes, their osteogenic differentiation ability has been proven to be stronger than that of BM-MSCs. 156 The most interesting characteristic of MSCs is their immunoregulatory functions, which are speculated to be related to either cell-to-cell contact or growth factor and cytokine secretion in response to environmental/microenvironmental stimuli. MSCs from different sources almost completely inhibit the proliferation of peripheral blood mononuclear cells (PBMCs) at PBMC:MSC ratios of 1:1 and 10:1. 149 At a higher ratio, BM-MSCs showed a significantly higher inhibitory effect than AT- or UC-MSCs. 153 Direct analysis of the immunosuppressive effects of BM- and UC-MSCs has revealed that these cells exert similar inhibitory effects in vitro with different mechanisms involved. 157 With these conflicting data, the mechanism of action related to the immune response of MSCs from different sources is still poorly understood, and long-term investigations both in preclinical studies and in clinical trial settings are needed to shed light on this complex immunomodulation function.

The great concern in MSC-based therapy is the fate of these cells post administration, especially through different delivery routes, including systemic administration via an intravenous (IV) route or tissue-specific administration, such as dorsal pancreatic administration. It is important to understand the distribution of these cells after injection to expand our understanding of the underlying mechanisms of action of treatments; in addition, this knowledge is required by authorized bodies (the Food and Drug Administration (FDA) in the United States or the regulation of advanced-therapy medicinal products in Europe, No. 1394/2007) prior to using these cells in clinical trials. The preclinical data using various labeling techniques provide important information demonstrating that MSCs do not have unwanted homing that could lead to the incorrect differentiation of the cells or inappropriate tumor formation. In a mouse model, human BM-MSCs and AT-MSCs delivered via an IV route are rapidly trapped in the lungs and then recirculate through the body after the first embolization process, with a small number of infused cells found mainly in the liver after the second embolization. 158 Using the technetium-99 m labeling method, intravenously infused human cells showed long-term persistence up to 13 months in the bone, BM compartment, spleen, muscle, and cartilage. 159 A similar result was reported in baboons, confirming the long-term homing of human MSCs in various tissues post administration. 160 Although the retainment of MSCs in the lungs might potentially reduce their systemic therapeutic effects, 161 it provides a strong advantage when these cells are used in the treatment of respiratory diseases. Local injection of MSCs also revealed their tissue-specific homing, as an injection of MSCs via the renal artery route resulted in the majority of the injected cells being found in the renal cortex. 162 Numerous studies have been conducted to track the migration of administered MSCs in human subjects. Henriksson and his team used MSCs labeled with iron sucrose in the treatment of intervertebral disc degeneration. 163 Their study showed that chondrocytes differentiated from infused MSCs could be detected at the injured intervertebral discs at 8 months but not at 28 months. A study conducted in a patient with hemophilia A using In-oxine-labeled MSCs showed that the majority of the cells were trapped in the lungs and liver 1 h post administration, followed by a reduction in the lungs and an increase in the number of cells in the liver after 6 days. 164 Interestingly, a small proportion of infused MSCs were found in the hemarthrosis site at the right ankle after 24 h, suggesting that MSCs are attracted and migrate to the injured site. The distribution of MSCs was also reported in the treatment of 21 patients diagnosed with type 2 diabetes using 18-FDG-tagged MSCs and visualized using positron emission tomography (PET). 165 The results illustrated that local delivery of MSCs via an intraarterial route is more effective than delivery via an IV route, as MSCs home to the pancreatic head (pancreaticoduodenal artery) or body (splenic artery). Therefore, although the available data related to the biodistribution of infused MSCs are still limited, the results obtained from both preclinical and clinical studies illustrate a comparable set of data supporting results on homing, migration to the injured site, and the major organs where infused MSCs are located. The following comprehensive and interesting reviews are highly recommended. 166 – 168

To date, 1426 registered clinical trials spanning different trial phases have used MSCs for therapeutic purposes, which is four times the number reported in 2013. 169 , 170 As supported by a large body of preclinical studies and advancements in conducting clinical trials, MSCs have been proven to be effective in the treatment of numerous diseases, including nervous system and brain disorders, pulmonary diseases, 171 cardiovascular conditions, 172 wound healing, etc. The outcomes of MSC-based therapy have been the subject of many intensive reviews and systematic analyses with the solid conclusion that these cells exhibit strong safety profiles and positive outcomes in most tested conditions. 173 – 175 In addition, the available data have revealed several potential mechanisms that could explain the beneficial effects of MSCs, including their homing efficiency, differentiation potential, production of trophic factors (including cytokines, chemokines, and growth factors), and immunomodulatory abilities. However, it is still not known which MSC types should be used for which diseases, as it seems to be that MSCs exhibit beneficial effects regardless of their sources. 169

Acquired brain and spinal cord injury treatment: BM-MSCs have emerged as key players

The theory that brain cells can never regenerate has been challenged by the discovery of newly formed neurons in the human adult hippocampus or the migration of stem cells in the brain in animal models. 176 These observations have triggered hope for regeneration in the context of neuronal diseases by using exogenous stem cell sources to replenish or boost the stem cell population in the brain. Moreover, the limited regenerative capacity of the brain and spinal cord is an obstacle for traditional treatments of neurodegenerative diseases, such as autism, cerebral palsy, stroke, and spinal cord injury (SCI). As current treatments cannot halt the progression of these diseases, studies throughout the world have sought to exploit cell-based therapies to treat neurodegenerative diseases on the basis of advances in the understanding and development of stem cell technology, including the use of MSCs. Successful stem cell therapy for treating brain disease requires therapeutic cells to reach the injured sites, where they can repair, replace, or at least prevent the deteriorative effects of neuronal damage. 177 Hence, the gold standard of cell-based therapy is to deliver the cells to the target site, stimulate the tissue repair machinery, and regulate immunological responses via either cell-to-cell contact or paracrine effects. 178 Among 315 registered clinical trials using stem cells for the treatment of brain diseases, MSCs and hematopoietic stem cells (HSCs; CD34+ cells isolated from either BM aspirate or UC blood) are the two main cell types investigated, whereas approximately 102 clinical trials used MSCs and 62 trials used HSCs for the treatment of brain disease (main search data from clinicaltrial.gov). MSCs are widely used in almost all clinical trials targeting different neuronal diseases, including multiple sclerosis, 179 stroke, 180 SCI, 181 cerebral palsy, 182 hypoxic-ischemic encephalopathy, 183 autism, 184 Parkinson’s disease, 185 Alzheimer’s disease 185 and ataxia. Among these trials in which MSCs were the major cells used, nearly two-thirds were for stroke, SCI, or multiple sclerosis. MSCs have been widely used in 29 registered clinical trials for stroke, with BM-MSCs being used in 16 of these trials. With 26 registered clinical trials, SCI is the second most common indication for using MSCs, with 16 of these trials using mainly expanded BM-MSCs. For multiple sclerosis, 15 trials employed BM-MSCs among a total of 23 trials conducted for the treatment of this disease. Hence, it is important to note that in neuronal diseases and disorders, BM-MSCs have emerged as the most commonly used therapeutic cells among other MSCs, such as AT-MSCs and UC-MSCs.

The outcomes of the use of BM-MSCs in the treatment of neuronal diseases have been widely reported in various clinical trial types. A review by Zheng et al. indicated that although the treatments appeared to be safe in patients diagnosed with stroke, there is a need for well-designed phase II multicentre studies to confirm the outcomes. 173 One of the earliest trials using autologous BM-MSCs was conducted by Bang et al. in five patients diagnosed with stroke in 2005. The results supported the safety and showed an improved Barthel index (BI) in MSC-treated patients. 186 In a 2-year follow-up clinical trial, 16 patients with stroke received BM-MSC infusions, and the results showed that the treatment was safe and improved clinical outcomes, such as motor impairment scale scores. 187 A study conducted in 12 patients with ischemic stroke showed that autologous BM-MSCs expanded in vitro using autologous serum improved the patient’s modified Rankin Scale (mRS), with a mean lesion volume reduced by 20% at 1 week post cell infusion. 188 In 2011, a modest increase in the Fugl Meyer and modified BI scores was observed after autologous administration of BM-MSCs in patients with chronic stroke. 189 More recently, a prospective, open-label, randomized controlled trial with blinded outcome evaluation was conducted, with 39 patients and 15 patients in the BM-MSC administration and control groups, respectively. The results of this study indicated that autologous BM-MSCs with autologous serum administration were safe, but the treatment led to no improvements at 3 months in modified Rankin Scale (mRS) scores, although leg motor improvement was observed. 180 Researchers explored whether the efficacy of BM-MSC administration was maintained over time in a 5-year follow-up clinical trial. Patients (85) were randomly assigned to either the MSC group or the control group, and follow-ups on safety and efficacy were performed for 5 years, with 52 patients being examined at the end of the study. The MSC group exhibited a significant improvement in terms of decreased mRS scores, whereas the number of patients with an mRS score increase of 0–3 was statistically significant. 187 Although autologous BM-MSCs did not improve the Basel index, mRS, or National Institutes of Health Stroke Scale (NIHSS) score 2 years post infusion, patients who received BM-MSC therapy showed improvement in their motor function score. 190 In addition, a prospective, open-label, randomized controlled trial by Lee et al. showed that autologous BM-MSCs primed with autologous “ischemic” serum significantly improved motor functions in the MSC-treated group. Neuroimaging analysis also illustrated a significant increase in interhemispheric connectivity and ipsilesional connectivity in the MSC group. 191 Recently, a single intravenous infection of allogeneic BM-MSCs has been proven to be safe and feasible in patients with chronic stroke with a significant improvement in BI score and NIHSS score. 192

In two systematic reviews using MSCs for the treatment of SCI, BM-MSCs ( n  = 16) and UC-MSCs ( n  = 5) were reported to be safe and well-tolerated. 193 , 194 The results indicated significant improvements in the stem cell administration groups compared with the control groups in terms of a composite of the American Spinal Injury Association (ASIA) impairment scale (AIS) grade, AIS grade A, and ASIA sensory scores and bladder function (Table ​ (Table1). 1 ). However, larger experimental groups with a randomized and multicentre design are needed for further confirmation of these findings. For multiple sclerosis, several early-phase (phase I/II) registered clinical studies have used BM-MSCs. A study compared the potential efficacy of BM-MSC and BM mononuclear cell (BMMNC) transplantation in 105 patients with spastic cerebral palsy. 195 The results showed that the GMFM (gross motor function measure) and the FMFM (fine motor function measure) scores of the BM-MSC transplant group were higher than those of the BMNNC transplant group at 3, 6, and 12 months of assessment. In terms of autism spectrum disorder, a review of 254 children after BMMNC transplantation found that over 90% of patients’ ISAA (Indian Scale for Assessment of Autism) and CARS (Childhood Autism Rating Scale) scores improved. Young patients and those in whom autism spectrum disorder was detected early generally showed better improvement. 196

The reported clinical trials using MSCs from AT, BM, and UC in the treatment of brain-related injuries and neurological disorders

25 WFT Timed 25‐Foot Walk, 9-PHT 9 Hole Peg Test, ADL Activities of Daily Living, AIS American Spinal Cord Injury Association (ASIA) Impairment Scale, CARS Childhood Autism Rating Scale CFA Comprehensive functional assessment, EDSS The Expanded Disability Status Scale, EMG electromyography, FMFM Fine motor function measurement, FOXP3 forkhead box P3, also known as scurfin, GMFM Gross motor function measurement, GMFM-88 Gross motor function measurement-88, IANR-SCIFRS International Association of Neurorestoratology-Spinal Cord Injury Functional Rating Scale, ISAA Indian Scale for Assessment of Autism, MMSE Mini‐Mental Status Examination, MRI Magnetic Resonance Imaging, mRS modified Rankin Scale, NCV nerve conduction velocity, NIHSS National Institutes of Health Stroke Scale, OCT Optical Coherence Tomography, SCI Spinal Cord Injury, SER somatosensory evoked potentials, VEP Visual Evoked Potential

One of the biggest limitations when using BM-MSCs is the bone marrow aspiration process, as it is an invasive procedure that can introduce a risk of complications, especially in pediatric and elderly patients. 197 Therefore, UC-MSCs have been suggested as an alternative to BM-MSCs and are being studied in clinical trials for the treatment of neurological diseases in approximately 1550 patients throughout the world; however, only three studies have been completed, with data published recently. 198 A recent study showed that UC-MSC administration improved both gross motor function and cognitive skills, assessed using the Activities of Daily Living (ADL), Comprehensive Function Assessment (CFA), and GMFM, in patients diagnosed with cerebral palsy. The improvements peaked 6 months post administration and lasted for 12 months after the first transplantation. 199 In a single-targeted phase I/II clinical trial using UC-MSCs for the treatment of autism, Riordan et al. reported decreases in Autism Treatment Evaluation Checklist (ATEC) and CARS scores for eight patients, but the paper has been retracted due to a violation of the journal’s guidelines. 200 In an open-label, phase I study, UC-MSCs were used as the main cells to treat 12 patients with autism spectrum disorder via IV infusions. It is important to note that five participants developed new class I anti-human leukocyte antigen in response to the specific lot of manufactured UC-MSCs, although these responses did not exhibit any immunological response or clinical manifestations. Only 50% of participants showed improvements in at least two autism-specific measurements. 201 Although not as widely used as BM-MSCs, these trials have demonstrated the efficacy of using UC-MSCs in the treatment of SCIs. In a pilot clinical study, Yang et al. showed that the use of UC-MSCs has the potential to improve disease status through an increase in total ASIA and SCI Functional Rating Scale of the International Association of Neurorestoratology (IANR-SCIFRS) scores, as well as an improvement in pinprick, light touch, motor and sphincter scores. 202 A study of 22 patients with SCIs showed a potential therapeutic effect in 13 patients post UC-MSC infusion. 203 AT-MSCs were also used to treat SCI, with a single case report indicating an improvement in neurological and motor functions in a domestic ferret patient. 204 However, a result obtained from another phase I trial using AT-MSCs showed mild improvements in neurological function in a small number of patients. 205 A phase II, randomized, double-blind, placebo-controlled, single-center, pilot clinical trial using AT-MSCs in the treatment of acute ischemic stroke published a data set that supports the safety of the therapy, although patients who received AT-MSCs showed a nonsignificant improvement after 24 months of follow-up. 206 In all of the above studies, the safety of using either AT-MSCs or UC-MSCs was evaluated, and no significant reactions were reported after infusion.

Therefore, based on the number of recovered patients post-transplantation and the number of recruited patients in large-scale trials using BM-MSCs, it seems that BM-MSCs are the prominent cells in regard to treating neurodegenerative disease with potentially good outcomes (Table ​ (Table1). 1 ). It is important to note that we do not negate the fact that AT- and UC-MSCs also show positive outcomes in the treatment of neuronal diseases, with numerous ongoing large-scale, multicentre, randomized, and placebo-control trials, 207 , 208 but we suggest alternative and thoughtful decisions regarding which sources of MSCs are best for the treatment of neuronal diseases and degenerative disorders.

Respiratory disease and lung fibrosis: clinical data support UC as a good source of MSCs

In the last decade, significant increases in respiratory disease incidence due to air pollution, smoking behavior, population aging, and recently, respiratory virus infections such as coronavirus disease 2019 (COVID-19) 209 have been observed, leading to substantial burdens on public health and healthcare systems worldwide. Respiratory inflammatory diseases, including bronchopulmonary dysplasia (BPD), chronic obstructive pulmonary disease (COPD), and acute respiratory distress syndrome (ARDS), have recently emerged as three prevalent pulmonary diseases in children and adults. These conditions are usually associated with inflammatory cell infiltration, a disruption of alveolar structural integrity, a reduction in alveolar fluid clearance ability, cytokine release and associated cytokine storms, airway remodeling, and the development of pulmonary fibrosis. Traditional treatments are focused on relieving symptoms and preventing disease progression using surfactants, artificial respiratory support, mechanical ventilation, and antibiotic/anti-inflammatory drugs, with limited effects on the damaged airway, alveolar fluid clearance, and other detrimental effects caused by the inflammatory response. MSCs are known for their immunomodulatory abilities, showing potential in injury reduction and aiding lung recovery after injury. According to ClinicalTrials.gov, from 2017 to date, there have been 159 studies testing the application of MSCs in the treatment of pulmonary diseases, including but not limited to BPD, COPD, and ARDS, suggesting a trend in the use of MSCs as an alternative approach for the treatment of respiratory diseases, especially MSCs from UC as an “off-the-shelf” and allogeneic source.

Extremely premature infants are born with arrested lung development at the canalicular-saccular phases prior to alveolarization and before pulmonary maturation occurs, which results in the development of BPD. 210 These infants require intensive care during the first three months of life using postnatal interventions, including positive pressure mechanical ventilation, external oxygen support, and surfactant infusions, and the newborns have recurrent infections that further compromise normal lung development. 211 To date, 13 clinical trials have been proposed to use UC-MSCs in the treatment of BPD, recruiting ~566 premature infants throughout the world, including Vietnam, Korea, the United States, Spain, Australia, and China. The majority of these trials use UC-derived stem cells for phases I and II, focusing on evaluating the safety and efficacy of stem cell-based therapy. 212 Human UC tissue and its derivative components are considered the most attractive cell sources for MSCs in the treatment of BPD due to the ease of obtaining them, being readily available, with no ethical concerns, low antigenicity, a high cell proliferation rate, and superior regenerative potential. Chang et al. used MSCs derived from UC blood in a phase I dose-escalation clinical trial to treat 9 preterm infants via intratracheal administration to prevent the development of BPD. 213 All 9 preterm infants survived, and only three developed BPD; these infants had significantly decreased BPD severity compared with the historically matched control group. A follow-up study of the same patients after 24 months indicated that only one infant had an E. cloacae infection after discharge at 4 months, with subsequent disseminated intravascular coagulation, which was later proven to be unrelated to the intervention. The remaining eight patients survived with normal pulmonary development and function, suggesting that the therapy was safe. MSCs from UC blood were also used for the treatment of 12 extremely low birthweight preterm patients using the same administration route, which further confirmed the safety of the therapy in the treatment of BPD, although ten of 12 infants still developed severe BPD at 36 weeks. 214 Our group also reported the safety and potential efficacy of using UC-MSCs in the treatment of four preterm infants, and the results supported the safety of UC-MSCs and demonstrated that patients could be weaned from oxygen supply and develop normal lung structure and function. 215 A phase II clinical trial of 66 infants born at 23–28 weeks with a birthweight of 500–1250 g who were recruited and randomized into an MSC-administration group and a control group was conducted. Although the results supported the safety of MSC administration in preterm infants, the efficacy of the treatment was not supported by statistical analysis, potentially due to the small sample size. Subgroup analysis showed that patients with severe BPD born at 23–24 weeks showed a significant improvement in BPD severity, but those born at 25–28 weeks did not. 216 Hence, it is important to conduct controlled phase II clinical trials with larger cohort sizes to further substantiate the efficacy of UC blood-derived MSCs in the treatment of infants with BPD.

With more than 65 million patients worldwide, COPD was the third-leading cause of death in 2020, according to World Health Organization records. COPD is classified as a chronic inflammatory and destructive pulmonary disease characterized by a progressive reduction in lung function. Averyanov et al. performed a randomized, placebo-controlled phase I/IIa study in 20 patients with mild to moderate idiopathic pulmonary fibrosis (IPF). Treatment group patients received two IV doses of allogeneic MSCs (2 × 10 8  cells) every 3 months, and the second group received a placebo. 217 Evaluation tests were performed at weeks 13, 26, 39, and 52. The 6-min walking test distance (6MWTD) results showed that patient fitness improved from week 13 onwards and was maintained until up to the 52nd week. Pulmonary function indicators improved markedly before and after treatment in the treated group but did not change significantly in the placebo group. The goal of MSC therapy in the treatment of COPD is to promote the regeneration of parenchymal cells and alveolar structure and the restoration of lung function. Based on the results of a phase I trial of a commercial BM-MSC product, Prochymal TM , which led to improvements in pulmonary function in treated patients, a multicentre, double-blind, placebo-controlled phase II trial was conducted in 62 patients diagnosed with COPD to determine the safety and potential efficacy of the product. Although the results supported the safety of BM-MSCs, their effectiveness in the treatment of COPD was not assured. No statistically significant differences in FEV 1 or FEV 1% , total lung capacity, or carbon monoxide diffusing capacity were detected after 2 years of follow-up between the two treatment groups. To date, there have been five clinical trials using BM-MSCs as the main stem cells for the treatment of COPD, but the overall clinical outcomes did not demonstrate the potential therapeutic effects of the treatment. 218 – 222 In clinical trial NCT001110252, the results showed that there was an overall reduction in the process of COPD pathological development 3 years after the administration of BM-MSCs, although the trial had a phase I design, with no control group, and evaluated only a small cohort (four patients). 219 To alleviate local inflammatory progression in COPD, Oliveira et al. studied the combination treatment of one-way endobronchial valve (EBV) and BM-MSC intubation. 223 Ten GOLD (Global Initiative for Obstructive Lung Disease) stage C or D patients were equally divided into 2 groups: one group received a dose of 10 8 cells before valve insertion, and the other group received a normal saline infusion. The follow-up time was 90 days. Inflammation was significantly improved as assessed by the CRP (C-reactive protein) index at 30 and 90 days after infusion. In addition, improvements in St. George’s Respiratory Questionnaire (SGRQ) scores indicated improved patient quality of life. Furthermore, an investigation into the homing ability of MSCs in vivo was performed on 9 GOLD patients, from stage A to stage D. Each patient received two 2 × 10 6 BM-MSC/kg IV infusions 1-week apart. 224 The marking of MSCs with indium-111 showed that MSCs were retained in the pulmonary vasculature longer in patients with mild COPD and that the levels of inflammatory mediators improved after 7 days of treatment. The results of the evaluation survey conducted after 1 year showed that the number of COPD exacerbations decreased to six times/year compared to 11 times/year before treatment. In addition, AT-MSCs present in the stromal vascular fraction were used to treat patients with COPD, and no adverse events were observed after 12 months of follow-up, but the clinical improvements post administration were not clear. 225 The results from a phase I clinical trial using AT-MSCs in eight patients with COPD also reported no significant change in pulmonary function test parameters. 226 A study evaluating the use of AT-MSCs as adjunctive therapy for COPD in 12 patients was performed. 227 AT was obtained using standard liposuction, MSCs were isolated, and 150–300 million cells were intravenously infused. The patients showed improvements in quality of life, with improved SGRQ scores after 3 and 6 months of treatment. Recently, UC-MSCs have emerged as potential allogeneic stem cell candidates for the treatment of COPD. 228 In a pilot clinical study, it was demonstrated that allogeneic administration of UC-MSCs in the treatment of COPD was safe and potentially effective. 229 In one study, 20 patients, including 9 at stage C and 11 at stage D per the GOLD classification, with histories of smoking were recruited and received cell-based therapy. The patients who received UC-MSC treatment showed significant reductions in Modified Medical Research Council scores, COPD assessment test scores, and the number of pulmonary exacerbations 6 months post administration. The results of the second trial using UC-MSCs showed that the mean FEV 1 /FVC ratios were increased along with improvements in SGRQ scores and 6MWTDs at three months post administration. 230 Although thorough assessments of the effectiveness of UC-MSCs are still in the early stages, the number of trials using UC-MSCs for the treatment of COPD is increasing steadily, with larger sample sizes and stronger designs (randomized or matched case–control studies), providing a data set strongly supporting the future applications of UC-MSCs. 231

The ongoing pandemic of the 21st century, the COVID-19 pandemic, emerged as a major pulmonary health problem worldwide, with a relatively high mortality rate. Numerous studies, reviews, and systematic analyses have been conducted to discuss and expand our knowledge of the virus and propose different mechanisms by which the virus could alter the immune system. 232 One of the most critical mechanisms is the generation of cytokine storms, which result from the initiation of hyperreactions of the adaptive immune response to viral infection. 233 These cytokine storms are formed by the establishment of waves of hypercytokinaemia generated from overreactive immune cells, which enhance their expression of TNF-α, IL-6, and IL-10, preventing T-lymphocyte recruitment and proliferation and culminating in T-lymphocyte apoptosis and T-cell exhaustion. In COVID-19, once a cytokine storm is formed, it spreads from an initial focal area through the body via circulation, which has been discussed in a comprehensive review by Jamilloux et al. 234 At the time of writing this review, there were 74 clinical trials using MSCs from UC (29 trials; including WJ-derived MSCs (WJ-MSCs) and placenta-derived MSCs (PL-MSCs)), AT (15 trials), and BM (11 trials) (comprehensive review 171 , 235 ). Hence, UC-MSCs have emerged as the most common MSCs for the treatment of COVID-19, with a total of 1047 patients participating in these trials. Among these trials, 15 completed trials using UC-MSCs (including WJ- and PL-MSCs) have been reported, with clinical data from approximately 600 recruited patients. 232 Eight of these 15 studies used allogenic UC-MSC transplantation to treat critically ill patients. 236 A list of case reports using UC-MSCs showed that the treatments were safe and well-tolerated in 14 patients with COVID-19, with the primary outcomes including increased percentages and numbers of T cells, 237 , 238 improved respiratory and renal functions, 239 reductions in inflammatory biomarker levels, 240 and positive outcomes in the PaO 2 /FiO 2 ratio. 240 In a pilot study conducted in ten patients with severe COVID-19, a single dose of UC-MSCs was safe and improved clinical outcomes, although the study did not investigate whether multiple doses of UC-MSCs could further improve the outcomes. 241 Two trials without a control group were conducted in 47 patients, and the results indicated that UC-MSCs were safe and feasible for the treatment of patients with COVID-19. 235 , 242 A single-center, open-label, individually randomized, standard treatment-controlled trial was performed in 41 patients (12 patients assigned to the UC-MSC group), and the results showed that significant improvements in C-reactive protein levels, IL-6 levels, oxygen indices, and lymphocyte numbers were found in the MSC groups. Chest computed tomography (CT) illustrated significant reductions in lung inflammatory responses as reflected by CT findings, the number of lobes involved, and pulmonary consolidation. 238 In a phase I trial conducted in 18 hospitalized patients with COVID-19, UC-MSCs were administered via an IV route in nine patients (five patients with moderate COVID-19 and 4 patients with severe COVID-19) at days 0, 3, and 6, with no treatment-related adverse events or severe adverse events. 243 Only one patient in the UC-MSC group required mechanical ventilation, compared to four patients in the control group. However, the clinical outcomes, such as COVID-19 symptoms, laboratory test results, CT findings of lung damage, and pulmonary function test parameters, were improved in both groups. Interestingly, a 1-year follow-up of the same sample revealed that the patients who received UC-MSC administration improved in terms of whole-lung lesion volume compared to the control group. 244 Moreover, chest CT at 12 months showed significant regeneration of lung tissue in the MSC-administered groups, whereas lung fibrosis was found in all patients in the control group. This finding is of interest because it indicates that a long time is needed to detect the regenerative functions of MSC-based therapy, as the biological process to enhance lung tissue regeneration occurs relatively slowly and requires multiple steps. The effects of UC-MSCs in the attenuation and prevention of the development of cytokine storms were illustrated in an interventional, prospective, three-parallel arm study with two control arms conducted in 30 patients in moderate and critical clinical conditions. 245 The results indicated a significant decrease in proinflammatory cytokines (IFNγ, IL-6, IL-17A, IL-2, and IL-12) and an increase in anti-inflammatory cytokines (IL-10, IL-13, and IL-1ra), suggesting that UC-MSCs might participate in the prevention of cytokine storm development. Lanzoni et al. performed a double-blind, randomized, controlled trial and found that UC-MSC infusions significantly decreased cytokine levels at day 6 and improved survival in patients with COVID-19 with ARDS. In this trial, 24 patients were randomized and assigned 1:1 to receive either MSCs or placebo. 246 MSC treatment was associated with a significant improvement in the survival rate without serious adverse events. To date, other trials conducted using UC-MSCs as the main MSCs provide a solid data set on their safety and efficacy in preventing the development of cytokine storms, reducing the inflammatory response, improving pulmonary function, reducing intensive care unit (ICU) stay duration, enhancing lung tissue regeneration, and reducing lung fibrosis progression. 240 , 247 – 249 In two large cohort studies (phase I with 210 patients and phase II with 100 patients), the volume of lung lesions and solid component injuries of patients’ lungs were reduced significantly after the administration of UC-MSCs, 250 and clinical symptoms and inflammatory levels were improved. 251 Of the 26 reported clinical trials for the treatment of COVID-19 with MSCs, 1 study used AT-MSCs as the main MSCs. 236 Thirteen COVID-19 adult patients under invasive mechanical ventilation who had received previous antiviral and/or anti-inflammatory treatments (including steroids, lopinavir/ritonavir, hydroxychloroquine, and/or tocilizumab, among others) were treated with allogeneic AT-MSCs. With a mean follow-up time of 16 days after infusion, 9/13 patients’ clinical symptoms improved, and 7/13 patients were intubated. A decrease in inflammatory cytokines and an increase in immunoregulatory cells were also observed in patients, especially in the group of patients with overall clinical improvement. Although there is a lack of clinical efficacy data supporting the use of AT-MSCs in the treatment of patients with COVID-19, AT-MSCs are still potential candidates for inhibiting COVID-19 due to their high secretory activity, strong immune-modulatory effects, and homing ability. 252 – 254

For ARDS, in a phase IIa trial, 60 patients with moderate to severe disease were randomized into 2 groups. A group of 40 patients received a single infusion of BM-MSCs at a dose of 1 × 10 6 cells/kg body weight, and another 20 patients received a placebo. 255 After 6 and 24 h of infusion, the decrease in plasma inflammatory cytokine levels in the MSC group was significantly greater than that in the placebo group. For severe pulmonary hypertension (PH) associated with BPD (BPD-PH), in a small trial, two preterm infants born at 26–27 weeks of age were intravenously administered heterologous BM-MSCs at a dose of 5 × 10 6 cells per kg of body weight; the treatment reduced oxygen requirements and supported respiration in the infants. 256 The administration of allogeneic AT-MSCs in the treatment of ARDS appeared to be safe and well-tolerated in 12 adult patients, but clinical outcomes were not observed. 257 The results of two patients who received BM-MSCs showed that both patients had improved respiratory function and hemodynamic function and a reduction in multiorgan failure. 258 Although the safety of BM-MSCs was confirmed in a multicentre, open-label, dose-escalation, phase I clinical trial (The Stem cells for ARDS treatment—START trial), 259 no significant improvements were found in a phase II trial, including in respiratory function and ARDS conditions. 260 The safety profile of UC-MSCs is also supported by the findings of a previous phase I clinical trial conducted in 9 patients, which showed that a single IV administration of UC-MSCs was safe and led to positive outcomes in terms of respiratory function and a reduction in the inflammatory response. 261 The findings of this study were also supported by those of the REALIST (Repair of Acute Respiratory Distress with Stromal Cell Administration) trial, which further confirmed the maximum tolerated dose of allogeneic UC-MSCs in patients with moderate to severe ARDS. 262

Although AT- and BM-MSCs have demonstrated therapeutic potential with similar mechanisms of action, UC-MSCs have emerged as potential candidates in the treatment of pulmonary diseases due to their ease of production as “off-the-shelf” products, rapid proliferation, noninvasive isolation methods, and supreme immunological regulation as well as anti-inflammatory effects. 263 However, it is important to note that there is a need to conduct phase III clinical trials with larger cohorts and trials with at least two sources of MSCs in the treatment of pulmonary conditions to further confirm this speculation. 264 Table ​ Table2 2 summarizes several clinical trials with published results discussed in this review.

The reported clinical trials using MSCs from AT, BM, and UC in the treatment of respiratory diseases

6MWD 6-min walk distance, ARDS acute respiratory distress syndrome, BPD bronchopulmonary dysplasia, COPD chronic obstructive pulmonary dysplasia, CRP C-reactive protein, CT computed tomography scan, DLCO diffusing capacity for CO, FCV flow-controlled ventilation, FEV 1 forced expiratory volume in 1 s, FiO 2 fraction of inspired oxygen, FVC forced vital capacity, PaO 2 partial pressure of oxygen, PEEP positive end-expiratory pressure, SGRQ St George’s Respiratory Questionnaire, TLC total lung capacity

Endocrine disorders, infertility/reproductive function recovery, and skin burns: should we consider AT-MSCs as the main MSCs based on their origin?

Endocrine disorders.

The human body maintains function and homeostatic regulation via a complex network of endocrine glands that synthesize and release a wide range of hormones. The endocrine system regulates body functions, including heartbeat, bone regeneration, sexual function, and metabolic activity. Endocrine system dysregulation plays a vital role in the development of diabetes, thyroid disease, growth disorder, sexual dysfunction, reproductive malfunction, and other metabolic disorders. The central dogma of regenerative medicine is the use of adult stem cells as a footprint for tissue regeneration and organ renewal. The functions of these stem cells are tightly regulated by microenvironmental stimuli from the nervous system (rapid response) and endocrine signals via hormones, growth factors, and cytokines. This harmonized and orchestrated system creates a symphony of signals that directly regulate tissue homeostasis and repair after injury. The disruption of these complex networks results in an imbalance of tissue homeostasis and regeneration that can lead to the development of endocrine disorders in humans, such as diabetes, sexual hormone deficiency, premature ovarian failure (POF), and Asherman syndrome.

In recent years, obesity and diabetes (type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM)) have been the two biggest challenges in endocrinology research, and the application of MSCs has emerged as a novel approach for therapeutic consideration. T1DM is characterized by the autoimmune destruction of pancreatic β-cells, whereas T2DM is defined as a combination of insulin resistance and pancreatic insulin-producing cell dysfunction. Regenerative medicine seeks to provide an exogenous cell source for replacing damaged or lost β-cells to achieve the goal of stabilizing patients’ blood glucose levels. To date, there are 28 clinical trials using MSCs in the treatment of T1DM ( http://www.clinicaltrials.gov , searched in October 2021), among which three trials were completed using autologous BM-MSCs ( {"type":"clinical-trial","attrs":{"text":"NCT01068951","term_id":"NCT01068951"}} NCT01068951 ), allogeneic BM-MSCs ( {"type":"clinical-trial","attrs":{"text":"NCT00690066","term_id":"NCT00690066"}} NCT00690066 ), and allogeneic AT-MSCs ( {"type":"clinical-trial","attrs":{"text":"NCT03920397","term_id":"NCT03920397"}} NCT03920397 ). Interestingly, UC-MSCs were the most favored MSCs for the remaining trials. All published studies confirmed the safety of MSC therapy in the treatment of T1DM with no adverse events. The first study using autologous BM-MSCs showed that patients who were randomized into the MSC-administration group showed an increase in C-peptide levels in response to a mixed-meal tolerance test (MMTT) in comparison to the control group. 265 Unfortunately, there was no significant improvement in C-peptide levels, HbA1 C or insulin requirements. The use of autologous AT-MSCs in combination with vitamin D was safe and improved HbA1 C levels 6 months post administration. 266 WJ-MSCs were used as the main MSCs for the treatment of new-onset T1DM, which showed a significant improvement in both HbA1 C and C-peptide levels when compared to those of the control group at three and six months post administration. 267 , 268 The combination of allogeneic WJ-MSCs with autologous BM-derived mononuclear cells improved insulin secretion and reduced insulin requirements in patients with T1DM. 269 In terms of T2DM, 23 studies were registered on clinicaltrials.gov (searched in October 2021), with six completed studies (three studies used BM-MSCs and three studies used allogeneic UC-MSCs). Although the number of studies using MSCs for the treatment of T2DM is small, their findings support the safety of MSCs, with no severe adverse events observed during the course of these studies. 270 It was confirmed that MSC therapy potentially reduced fasting blood glucose and HbA1 C levels and increased C-peptide levels. However, these effects were short-term, and multiple doses were required to maintain the MSC effects. Interestingly, the autologous MSC approach in the treatment of patients with diabetes in general is hampered, as both BM-MSCs and AT-MSCs isolated from patients with diabetes showed reduced stemness and functional characteristics. 271 , 272 In addition, the durations of diabetes and obesity are strongly associated with autologous BM-MSC metabolic function, especially mitochondrial respiration, and the accumulation of mitochondrial DNA, which directly interfere with the functions of BM-MSCs and reduce the effectiveness of the therapy. 271 Therefore, the allogeneic approach using MSCs from healthy donors provides an alternative approach for stem cell therapy in the treatment of patients with diabetes.

Infertility and reproductive function recovery

Modern society is increasingly facing the problem of infertility, which is defined as the inability to become pregnant after more than 1 year of unprotected intercourse. 273 This problem has emerged as an important worldwide health issue and social burden. Assisted reproductive techniques and in vitro fertilization technology have recently become the most effective methods for the treatment of infertility in humans, but the use of these approaches is limited, as they cannot be applied in patients with no sperm or those who are unable to support implantation during pregnancy, they are associated with complications, they are time-consuming and expensive, and they are associated with ethical issues in certain territories. 274 Numerous conditions are related to infertility, including POF, nonobstructive azoospermia, endometrial dysfunction, and Asherman syndrome. Recent progress has been illustrated in preclinical studies for the potential applications of stem cell-based therapy for reproductive function recovery, especially recent studies in the field of MSCs, which provide new hope for patients with infertility and reproductive disorders. 275

POF is characterized by a loss of ovarian activity during middle age (before 40 years old) and affects 1–2% of women of reproductive age. 276 Patients diagnosed with POF exhibit oligo-/amenorrhea for at least 4 months, with increased levels of follicle-stimulating hormone (FSH) (>25 IU/L) on two occasions more than 1 month apart. 277 Diverse factors, such as genetic backgrounds, autoimmune disorders, environmental conditions, and iatrogenic and idiopathic situations, have been reported to be the cause of POF. 278 POF can be treated with limited effectiveness via psychosocial support, hormone replacement intervention, and fertility management. 279 MSCs from AT, BM, and UC have been used in the treatment of POF, with improvements in ovarian function in preclinical studies using chemotherapy-induced POF animal models. The early published POF study using BM-MSCs as the main cell source is a single case report in which a perimenopausal woman showed an improvement in follicular regeneration, and increased AMH levels resulted in a successful pregnancy followed by delivery of a healthy infant. 280 A report using autologous BM-MSCs in two women with POF illustrated an increase in baseline estrogen levels and the volume of the treated ovaries along with amelioration of menopausal symptoms. 281 The clinical procedures used in this early trial were invasive, as patients underwent two operations: (1) BM aspiration and (2) laparoscopy. A similar approach was used in two trials conducted in 10 women with POF (age range from 26–33 years old) and 30 patients (age from 18 to 40 years old). 282 A later study investigated two different routes of cell delivery, including laparoscopy and the ovarian artery, but the results have not been reported at this time. 282 Based on the positive outcomes of the mouse model, an autologous stem cell ovarian transplantation (ASCOT) trial was deployed using BM-derived stem cells with encouraging observations of improved ovarian function, as determined by elevated levels of AMH and AFC in 81.3% of participants, six pregnancies, and the successful delivery of three healthy babies. 283 A randomized trial ( {"type":"clinical-trial","attrs":{"text":"NCT03535480","term_id":"NCT03535480"}} NCT03535480 ) was conducted in 20 patients with POF aged less than 39 years to further elaborate on the results of the ASCOT trial. 284 To date, there are no completed trials using AT-MSCs or UC-MSCs in the treatment of patients with POF, limiting the evaluation of these MSCs in the treatment of POF. The speculated reason is that POF is a rare disease, affecting 1% of women younger than 40 years, and with improvements in assisted productive technology, patients have several alternative options to enhance the recovery of reproductive function. 285

Wound healing and skin burns

Burns are the fourth most common injury worldwide, affecting ~11 million people, and are a major cause of death (180,000 patients annually). The severity of burns is defined based on the percentage of surface area burned, burn depth, burn location and patient age, and burns are usually classified into first-, second-, third-, and fourth-degree burns on the basis of their severity. 286 Postburn recovery depends on the severity of the burn and the effectiveness of treatment. Rapid healing may occur over weeks, while alternatively, healing can take months, with the ultimate result being scar formation and disability in patients with severe burns. Different from mechanical injury, burn injury is an invasive progression of damage to tissue at the burn site, including both mechanical damage to the skin surface and biological damage caused by natural apoptosis that prolongs excessive inflammation, oxidative stress, and impaired tissue perfusion. 287 To date, completely reversing the devastating damage of severe burns remains unachievable in medicine, and stem cell therapy provides an alternative option for patients with burn injury. The first case report of the use of BM-MSCs to treat a 45-year-old patient with burns on 40% of their body demonstrated the safety of the therapy and showed partial improvements in vascularization at the wound site and reduced coarse cicatrices. 288 , 289 Later, patients with second- and third-degree burns as well as deep burns were treated using either autologous BM-MSCs or allogeneic BM-MSCs by spraying the MSCs onto the burn sites or adding MSCs over a dermal matrix sheet to cover the wound. The results in these case reports revealed the potential efficacy of MSC-based therapy, which not only enhanced the speed of wound recovery but also reduced pain and improved blood supply without introducing infection. 288 , 290 , 291 In 2017, a study conducted in 60 patients with 10–25% of their total body surface areas burned treated with either autologous BM-MSCs or UC-MSCs showed that both MSC types improved the rate of healing and reduced the hospitalization period. 292 The drawback of BM-MSCs in the treatment of burns is the invasive harvesting method, which causes pain and possible complications in patients. Hence, treatment with allogeneic MSCs obtained from healthy donors is the method of choice, and AT- and UC-MSCs are two suitable candidates for this option. To date, a limited number of clinical trials have been conducted using MSC therapy. These trials have several limitations in trial design, such as a lack of a negative control group and blinding, small sample sizes, and the use of standardized measurement tools for burn injury and wound healing. Currently, AT-MSCs are being used in seven ongoing phase I and II trials in the treatment of burns. Hence, it is important to note that among the most widely studied MSCs, AT-MSCs have advantages over BM-MSCs when obtained from an allogeneic source, while their abilities in burn treatment remain to be determined. The main MSCs that should be used in the regeneration of burn tissue remain undefined (Table ​ (Table3), 3 ), and we observed the trend that AT-MSCs are more suitable candidates due to their biological nature, which contributes to the generation of keratinocytes and secretion profiles that strongly enhance the skin regeneration process. 293 – 296

The reported clinical trials using MSCs from AT, BM, and UC in the treatment of the endocrinological disorder, reproductive disease, and skin healing

AFC antral follicle count, AMH anti-Müllerian hormone, AUC area under the curve (oral glucose tolerance test), FSH follicle-stimulating hormone, HbA1C hemoglobin A1C, MMTT mixed-meal tolerance test

MSC applications in cardiovascular disease: a promising but still controversial field

In the last two decades, great advancements have been achieved in the development of novel regenerative medicine and cardiovascular research, especially stem cell technology. 297 The discovery of human embryonic stem cells and human induced pluripotent stem cells (hiPSCs) opened a new door for basic research and therapeutic investigation of the use of these cells to treat different diseases. 298 However, the clinical path of hiPSCs and hiPSC-derived cardiomyocytes in the treatment of cardiovascular diseases is limited due to the potential for teratoma formation with hiPSCs and the immaturity of hiPSC-derived cardiomyocytes, which might pose a risk of cancer formation, 299 arrhythmia, and cardiac arrest to patients. 300 A recently emerged stem cell type is adult stem cells/progenitor cells, including MSCs, which can stimulate myocardial repair post administration due to their paracrine effects. Promising results of MSC-based therapy obtained from preclinical studies of cardiac diseases enhance the knowledge and strengthen the clinical research to investigate the safety and efficacy in a clinical trial setting. There are papers that discuss the importance of MSC therapy in the treatment of cardiovascular diseases, with the following references being highly recommended. 301 – 306 To date, 36 trials have evaluated the therapeutic potential of MSCs in different pathological conditions, with the most prevalent types being BM-MSCs (25 trials), followed by UC-MSCs (7 trials) and AT-MSCs (4 trials). 303 However, the reported results are contradictory and create controversy about the efficacy of the treatments.

One of the first trials using MSCs in the treatment of chronic heart failure was the Cardiopoietic Stem Cell Therapy in Heart Failure (C-CURE) trial, a multicentre, randomized clinical trial that recruited 47 patients. The trial findings supported the safety of BM-MSC therapy and provided a data set that demonstrated improvements in cardiovascular scores along with New York Heart Association functional class, quality of life, and general physical health. 307 Despite these encouraging results in the phase I trial, the treatment failed to achieve the primary outcomes in the phase II/III trial (CHART-1 trial), including no significant improvements in cardiac structure or function or patient quality of life. 308 A positive outcome was also found in a phase I/II, randomized pilot study called the POSEIDON trial, which was the first trial to demonstrate the superior effectiveness of the administration of allogeneic BM-MSCs compared to allogeneic MSCs from other sources. 309 , 310 Published results from the MSC-HF study, with 4 years of follow-up results, 311 , 312 and the TRIDENT study 313 illustrated the positive outcomes of BM-MSCs in the treatment of heart failure. However, a contradictory result from the recently published CONCERT-HF trial demonstrated that the administration of autologous BM-MSCs to patients diagnosed with chronic ischemic heart failure did not improve left ventricular function or reduce scar size at 12 months post administration, but the patient’s quality of life was improved. 314 This observation is similar to that of the TAC-HFT trial 315 but completely different from the reported results of the MSC-HF trial. A comprehensive investigation is still needed to determine the reasons behind these contradictory results. The largest clinical trial to date using BM-MSCs is the DREAM-HF study, which was a randomized, double-blind, placebo-controlled, phase III trial that was conducted at 55 sites across North America and recruited a total of 565 patients with ischemic and nonischaemic heart failure. 172 Although recent reports from the sponsor confirmed that the trial missed its primary endpoint (a reduction in recurrent heart failure-related hospitalization), other prespecified endpoints were met, such as a reduction in overall major adverse cardiac events (including death, myocardial infarction, and stroke). 306 Thus, a complete report from the DREAM-HF trial will provide pivotal data supporting the therapeutic potential of BM-MSCs in the treatment of heart failure and open a new path for the FDA to approve cell-based therapy for cardiovascular diseases.

The early trial using AT-derived cells was the PRECISE trial, which was a phase I, randomized, placebo-controlled, double-blind study that examined the safety and efficacy of adipose-derived regenerative cells (ADRCs) in the treatment of chronic ischemic cardiomyopathy. 316 ADRCs are a homogenous population of cells obtained from the vascular stromal fraction of AT, which contains a small proportion of AT-MSCs. 317 Although the study supported the safety of ADRC administration and illustrated a preserved functional capacity (peak VO 2 ) in the treated group and improvements in heart wall motion, neither poor left ventricle (LV) volume nor poor left ventricular ejection fraction (LVEF) was ameliorated. The follow-up trial of the PRECISE trial, called the ATHENA trial, was conducted in 31 patients, although the study was terminated prematurely because two cerebrovascular events occurred, which were not related to the cell product itself. 318 The results of the study illustrated increases in functional capacity, hospitalization rate, and MLHFQ scores, but the LV volume and LVEF were not significantly different between the two groups. Kastrup and colleagues conducted the first in vitro expanded AT-MSC trial in ten patients with ischemic heart disease and ischemic heart failure in 2017. The results confirmed that ready-to-use AT-MSCs were well-tolerated and potentially effective in the treatment of ischemic heart disease and heart failure. 319 Comparable results of AT-MSCs were also reported from the MyStromalCell Trial, which was a randomized placebo-controlled study. In this trial, 61 patients were randomized at a 2:1 ratio into two groups, with the results showing no significant difference in the primary endpoint, which was a change in the maximal bicycle exercise tolerance test (ETT) score from baseline to 6 months post administration. 320 A 3-year follow-up report from the MyStromalCell Trial confirmed that patients who received AT-MSC administration maintained their preserved exercise capacity and their cardiac symptoms improved, whereas the control group experienced a significant reduction in exercise performance and a worsened cardiovascular condition. 321

UC-MSCs are potential allogeneic cells for the treatment of cardiovascular disease, as they are “ready to use” and easy to isolate, they rapidly proliferate, and they secrete hepatocyte growth factors, 322 which are involved in cardioprotection and cardiovascular regeneration. 323 The pilot study using UC-MSCs in 30 patients with heart failure, called the RIMECARD trial, was the first reported trial for which the results supported the effectiveness of UC-MSCs, as seen in the improved ejection fraction, left ventricular function, functional status, and quality of life in patients administered UC-MSCs. 324 Encouraging results reported from a phase I/II HUC-HEART trial 325 showed improvements in LVEF and reductions in the size of the injured area of the myocardium. However, the opposite observations were also reported from a recently published phase I randomized trial using a combination of UC-MSCs and a collagen scaffold in patients with ischemic heart conditions, in which the size of fibrotic scar tissue was not significantly reduced. 326

Although MSCs from AT, BM, and UC have proven to be safe and feasible in the treatment of cardiovascular diseases, the correlation between the MSC types and their therapeutic potentials is still uncertain because different results have been reported from different clinical trials (Table ​ (Table4). 4 ). The mechanisms by which MSCs participate in recovery and enhance myocardial regeneration have been discussed comprehensively in a recently published review; 305 , 327 therefore, they will not be discussed in this review. In fact, the challenges of MSC-based therapy in cardiovascular diseases have been clearly described previously, 328 including (1) the lack of an in vitro evaluation of the transdifferentiation potential of MSCs to functional cardiac and endothelial cells, 329 (2) the uncontrollable differentiation of MSCs to undesirable cell types post administration, 330 and (3) the undistinguishable nature of MSCs derived from different sources with various levels of differentiation potential. 331 Therefore, the applications of MSC-based therapy in cardiovascular disease are still in their immature stage, with potential benefits to patients. Thus, there is a need to conduct large-scale, well-designed randomized clinical trials not only to confirm the therapeutic potential of MSCs from various sources but also to enhance our knowledge of cardiovascular regeneration post administration.

The reported clinical trials using MSCs from AT, BM, and UC in the treatment of cardiovascular diseases

6MWT 6-min walk test, CCS Canadian Cardiovascular Society, ESV end-systolic volume, LVEDV left ventricular end-diastolic chamber volume, LVEF left ventricular ejection fraction, LVESV left ventricular end-systolic volume, MACE major adverse cardiovascular events, MET metabolic equivalents, MLHF Minnesota Living with Heart Failure, MLHFQ Minnesota Living with Heart Failure Questionnaire, MVO 2 maximal oxygen consumption, NYHA New York Heart Association, SPECT single photon emission computed tomography, TNF-α tumor necrosis factor alpha, WMSI wall motion score index

Proposed mechanism of BM-MSCs in the treatment of acquired brain and spinal injury

Bones are complex structures constituting a part of the vertebrate skeleton, and they play a vital role in the production of blood cells from HSCs. Similar to the functions of most vertebrate organs, bone function is tightly regulated by its constituents and by long-range signaling from AT and the adrenal glands, parathyroid glands, and nervous system. 332 The central nervous system (CNS) orchestrates the voluntary and involuntary input transmitted by a network of peripheral nerves, which act as the bridge between the nervous system and target organs. The CNS controls involuntary responses via the autonomic nervous system (ANS), consisting of the sympathetic nervous system and the parasympathetic nervous system, and voluntary responses via the somatic nervous system. The ANS penetrates deep into the BM cavity, reaching the regions of hematopoietic activity to deliver neurotransmitters that tightly regulate BM stem cell niches. 333 The BM microenvironment consists of various cell types that participate in the maintenance of HSC niches, which are composed of specialized cells, including BM-MSCs (Fig. ​ (Fig.3a). 3a ). The release of a specific neurotransmitter, circadian norepinephrine, from the sympathetic nervous system at nerve terminals leads to a reduction in the circadian expression of C–X-C chemokine ligand 12 (CXCL12, which is also known as stromal cell-derived factor-1 (SDF-1)) by Nestin + /NG2 2+ BM-MSCs, resulting in the secretion of HSCs into the peripheral bloodstream. 334 , 335 In fact, BM-MSCs play a significant role in the regulation of HSC quiescence and are closely associated with arterioles and sympathetic nervous system nerve fibers. Nestin-expressing BM-MSCs have been shown to express high levels of SDF-1, stem cell factor (SCF), angiopoietin-1 (Ang-1), interleukin-7, vascular cell adhesion molecule 1 (VCAM-1), and osteopontin (OPN), which are directly involved in the regulation and maintenance of HSC quiescence. 336 The depletion of BM-MSCs in BM leads to the mobilization of HSCs into the peripheral bloodstream and spleen. The findings from a previous study demonstrated that reduced SDF-1 expression in norepinephrine-treated BM-MSCs resulted in the mobilization of CXCR4 + HSCs into circulation. 337 The ability of BM-MSCs to produce SDF-1 is tightly related to their neuronal protective functions. 338 SDF-1 is a member of a chemokine subfamily that orchestrates an enormous diversity of pathways and functions in the CNS, such as neuronal survival and proliferation. The chemokine has two receptors, CXCR4 and CXCR7, that are involved in the pathogenic development of neurodegenerative and neuroinflammatory diseases. 339 In the damaged brain, SDF-1 functions as a stem cell homing signal, and in acquired immune deficiency syndrome (AIDS), SDF-1 has been reported to be involved in the protection of damaged neurons by preventing apoptosis. In a traumatic brain injury model, SDF-1 was found to function as an inhibitor of the caspase-3 pathway by upregulating the Bcl-2/Bax ratio, which in turn protects neurons from apoptosis. 340 Moreover, the release of SDF-1 also facilitates cell recruitment, cell migration, and the homing of neuronal precursor cells in the adult CNS by activating the CXCR4 receptor. 341 , 342 Existing data support that SDF-1 acts as the guiding signal for the regeneration of axon growth in damaged neurons and enhances spinal nerve regeneration. 343 , 344 Hence, the ability of BM-MSCs to express SDF-1 in response to the neuronal environment provides a unique neuronal protective effect that could explain the potential therapeutic efficacy of BM-MSCs in the treatment of neurodegenerative diseases (Fig. ​ (Fig.3b 3b ).

The nature of the “stem niche” of bone marrow-derived mesenchymal stem cells (BM-MSCs) supports their therapeutic potential in neuron-related diseases. a Bone marrow is a complex stem cell niche regulated directly by the central nervous system to maintain bone marrow homeostasis and haematopoietic stem cell (HSC) functions. MSCs in bone marrow respond to the environmental changes through the release of norepinephrine (NE) from the sympathetic nerves that regulate the synthesis of SDF-1 and the migration of HSCs through the sinusoids. The secretion of stem cell factors (SCFs), VCAM-1 and angiotensin-1 from MSCs also plays a significant role in the maintenance of HSCs. b BM-MSCs have the ability to produce and release SDF-1, which directly contributes to neuroprotective functions at the damaged site through interaction with its receptors CXCR4/7, located on the neuronal membrane. c Neuronal protection and the functional remyelination induced by BM-MSCs are also modulated by the release of a wide range of growth factors, including VEGF, BDNF, and NGF, by the BM-MSCs. d BM-MSCs also have the ability to regulate neuronal immune responses by direct interaction or paracrine communication with microglia. Figure was created with BioRender.com

The migration of exogenous MSCs after systemic administration to the brain is limited by the physical blood–brain barrier (BBB), which is a selective barrier formed by CNS endothelial cells to restrict the passage of molecules and cells. The mechanism of molecular movement across the BBB is well established, but how stem cells can bypass the BBB and home to the brain remains unclear. Recent studies have reported that MSCs are able to migrate through endothelial cell sheets by paracellular or transcellular transport followed by migration to the injured or inflammatory site of the brain. 345 , 346 During certain injuries or ischemic events, such as brain injury, stroke, or cerebral palsy, the integrity and efficiency of BBB protection is compromised, which allows MSC migration across the BBB via paracellular transport through the transient formation of interendothelial gaps. 347 CD24 expression has been detected in human BM-MSCs, which are regulated by TGF-β3, 348 allowing them to interact with activated endothelial cells via P-selectin and initiate the tethering and rolling steps of MSCs. 349 Additionally, BM-MSCs express high levels of CXCR4 or CXCR7, 350 , 351 which bind to integrin receptors, such as VLA-4, to activate the integrin-binding process and allow the cells to anchor to endothelial cells, followed by the migration of MSCs through the endothelial cell layer and basement membrane in a process called transmigration. 352 This process is facilitated by the secretion of matrix metalloproteinases (MMPs), which degrade the endothelial basement membrane, allowing BM-MSCs to enter the brain environment. 353 , 354 BM-MSCs can also regulate the integrity of the BBB via the secretion of tissue inhibitor of matrix metalloproteinase-3 (TIMP3), which has been shown to ameliorate the effects of a compromised BBB in traumatic brain injury. 355 The secretion of TIMP3 from MSCs directly blocked vascular endothelial growth factor a (VEGF-a)-induced breakdown of endothelial cell adherent junctions, demonstrating the potential mechanism of BM-MSCs in the regulation of BBB integrity.

The therapeutic applications of BM-MSCs in neurodegenerative conditions have been significantly increased by the demonstration of BM-MSC involvement in axonal and functional remyelination processes. Remyelination is a spontaneous regenerative process occurring in the human CNS to protect oligodendrocytes, neurons, and myelin sheaths from neuronal degenerative diseases. 356 Remyelination is considered a neuroprotective process that limits axonal degeneration by demyelination and neuronal damage. The first mechanism of action of BM-MSCs related to remyelination is the activation of the JAK/STAT3 pathway to regulate dorsal root ganglia development. 357 It was reported that BM-MSCs secrete vascular endothelial growth factor-A (VEGF-A), 358 brain-derived neurotrophic factor (BDNF), interleukin-6, and leukemia inhibitor factor (LIF), which directly function in neurogenesis and neurite growth. 357 VEGF-A is a key regulator of hemangiogenesis during development and bone homeostasis. Postnatally, osteoblast- and MSC-derived VEGF plays a critical role in maintaining and regulating bone homeostasis by stimulating MSC differentiation into osteoblasts and suppressing their adipogenic differentiation. 359 – 361 To balance osteoblast and adipogenic differentiation, VEGF forms a functional link with the nuclear envelope protein laminin A, which in turn directly regulates the osteoblast and adipocyte transcription factors Runx2 and PPARγ, respectively. 361 , 362 In the brain, VEGF is a potent growth factor mediating angiogenesis, neural migration, and neuroprotection. VEGF-A, secreted from BM-MSCs under in vitro xeno- and serum-free culture conditions, is the most studied member of the VEGF family and is suggested to play a protective role against cognitive impairment, such as in the context of Alzheimer’s disease pathology or stroke. 363 – 365 Recently, it was reported that the neurotrophic and neuroprotective function of VEGF is mediated through VEGFR2/Flk-1 receptors, which are expressed in the neuroproliferative zones and extend to astroglia and endothelial cells. 366 In animal models of intracerebral hemorrhage and cerebral ischemia, the transfusion of Flk-1-positive BM-MSCs promotes behavioral recovery and anti-inflammatory and angiogenic effects. 367 , 368 Moreover, supplementation with VEGF-A in neuronal disorders enhances intraneural angiogenesis, improves nerve regeneration, and promotes neurotrophic capacities, which in turn increase myelin thickness via the activation of the prosurvival transcription factor nuclear factor-kappa B (NF-kB). This activation, together with the downregulation of Mdm2 and increased expression of the pro-apoptotic transcription factor p53, is considered to be the neuroprotective process associated with an increased VEGF-A level. 369 – 371 An analysis of microRNA (miRNA) in extracellular vesicles (EVs) secreted from BM-MSCs revealed that BM-MSCs release substantial amounts of miRNA133b, which suppresses the expression of connective tissue growth factor (CTGF) and protects hippocampal neurons from apoptosis and inflammatory injury 372 – 374 (Fig. ​ (Fig.3c 3c ).

In terms of immunoregulatory functions, the administration of human BM-MSCs into immunocompetent mice subjected to SCI or brain ischemia showed that BM-MSCs exhibited a short-term neuronal protective function against neurological damage (Fig. ​ (Fig.3d). 3d ). Further investigation demonstrated the ability of BM-MSCs to directly communicate with host microglia/macrophages and convert them from phenotypic polarization into alternative activated microglia/macrophages (AAMs), which are key players in axonal extension and the reconstruction of neuronal networks. 375 Other studies have also illustrated that the administration of AAMs directly to the injured spinal cord induced axonal regrowth and functional improvement. 376 The mechanism by which BM-MSCs activate the conversion of microglia/macrophages occurs through two representative macrophage-related chemokine axes, CCL2/CCR2 and CCL-5/CCR5, both of which exhibit acute or chronic elevation following brain injury or SCI. 377 The CCL2/CCR2 axis contributed to the enhancement of inflammatory function, and BM-MSC-mediated induction of CCL2 did not alter the total granulocyte number (Fig. ​ (Fig.3d). 3d ). Although the chemokine-mediated mechanism of BM-MSCs in the activation of AAMs and enhanced axonal regeneration at the damage sites is evident, the direct mechanism by which the communication between BM-MSCs and the target cells results in these phenomena remains unclear, and further investigation is needed.

BM-MSCs also confer the ability to regulate the inflammatory regulation of the immune cells present in the brain by (1) promoting the polarization of macrophages toward the M2 type, (2) suppressing T-lymphocyte activities, (3) stimulating the proliferation and differentiation of regulatory T cells (Tregs), and (4) inhibiting the activation of natural killer (NK) cells. BM-MSCs secrete glial cell line-derived neurotrophic factor (GDNF), a specific growth factor that contributes directly to the transition of the microglial destructive M1 phenotype into the regenerative M2 phenotype during the neuroinflammatory process. 378 A similar result was also found in AT- 379 and UC-MSCs 380 under neuroinflammation-associated conditions, suggesting that AT-, BM-, and UC-MSCs share the same mechanism in promoting macrophage polarization. In terms of T-lymphocyte suppression, compared to MSCs from AT and BM, UC-MSCs show the strongest potential to inhibit the proliferation of T-lymphocytes by promoting cell cycle arrest (G0/G1 phase) and apoptosis. 381 In addition, UC-MSCs have been proven to be more effective in promoting the proliferation of Tregs 382 and inhibiting NK activation. 383 Although MSCs are well-known for their inflammatory regulatory ability, the mechanism is not exclusive to BM-MSCs, especially in neurological disorders. 384

Proposed mechanism of UC-MSCs in the treatment of pulmonary diseases and lung fibrosis

In contrast to AT-MSCs and BM-MSCs, UC-MSCs have lower expression of major histocompatibility complex I (MHC I) and no expression of MHC II, which prevents the complications of immune rejection. 385 Moreover, as UC is considered a waste product after birth, with the option of noninvasive collection, UC-MSCs are easier to obtain and culture than AD- and BM-MSCs. 386 These advantages of UC-MSCs have contributed to their use in the treatment of pulmonary diseases, especially during the rampant COVID-19 pandemic, as “off-the-shelf” products. Numerous pulmonary diseases have been the subject of applications of UC-MSCs, including BPD, COPD, ARDS, and COVID-19-induced ARDS. In BPD, premature infants are born before the alveolarization process, resulting in arrested lung development and alveolar maturation. Upon administration via an IV route, the majority of exogenous UC-MSCs reach the immature lung and directly interact with immune cells to exert their immunomodulatory properties via cell-to-cell interaction mechanisms (Fig. ​ (Fig.4a). 4a ). UC-MSCs interact with T cells via the PD-L1 ligand, which binds to the PD-1 inhibitory molecule on T cells, resulting in the suppression of CD3+ T-cell proliferation and effector T-cell responses. 387 In addition, UC-MSCs also express CD54 (ICAM-1), which plays a crucial role in the immunomodulatory functions of T cells. 388 Direct contact between UC-MSCs and macrophages via CD54 expression on UC-MSCs promotes the immune regulation of UC-MSCs via the regulation of phagocytosis by monocytes. 389 Moreover, the contact of UC-MSCs with macrophages during proinflammatory responses increases the secretion of TSG-6 by UC-MSCs, which in turn promotes the inhibitory regulation of CD3+ T cells, macrophages, and monocytes by MSCs. 390 Recently, upregulation of SDF-1 was described in neonatal lung injury, especially in layers of the respiratory epithelium. 391 SDF-1 has been shown to participate in the migration and initiation of the homing process of MSCs via the CXCR4 receptors on their surface. 392 It was reported that UC-MSCs express low levels of CXCR4, allowing them to induce SDF-1-associated migration processes via the Akt, ERK, and p38 signal transduction pathways. 393 Hence, in BPD, the upregulation of SDF-1 together with the homing ability of UC-MSCs strongly supports the therapeutic effects of UC-MSCs in the treatment of BPD. Furthermore, UC-MSCs have the ability to communicate with immune cells via cell-to-cell contact to reduce proinflammatory responses and the production of proinflammatory cytokines (such as TGF-β, INF-γ, macrophage MIF, and TNF-α). The modulation of the human innate immune system by UC-MSCs is mediated by cell–cell interactions via CD54-LFA-1 that switch macrophage polarization processes, promoting the proliferation of M2 macrophages, which in turn reduce inflammatory responses in the immature lung. 394 Moreover, UC-MSCs also have the ability to produce VEGF and hepatocyte growth factors (HGFs), promoting angiogenesis and enhancing lung maturation. 395

Adipose tissue-derived mesenchymal stem cells (AT-MSCs) and the nature of their tissue of origin support their use in therapeutic applications. a Adipose tissue is considered an endocrine organ, supporting and regulating various functions, including appetite regulation, immune regulation, sex hormone and glucocorticoid metabolism, energy production, the orchestration of reproduction, the control of vascularization, and blood flow, the regulation of coagulation, and angiogenesis and skin regeneration. b In terms of metabolic disorders, such as type 2 diabetes mellitus (T2DM), as adipose tissue is directly involved in the metabolism of glucose and lipids and the regulation of appetite, the detrimental effects of T2DM also alter the functions of AT-MSCs, which in turn, hampers their therapeutic effects. Hence, the use of autologous AT-MSCs is not recommended for the treatment of metabolic disorders, including T2DM, suggesting that allogeneic AT-MSCs from healthy donors could be a better alternative approach. c AT-MSCs are suitable for the treatment of reproductive disorders due to their unique ability to mobilize and home to the thecal layer of the injured ovary, enhance the regeneration and maturation of thecal cells, increase the structure and function of damaged ovaries via exosome-activated SMAD, decrease oxidative stress and autophagy, and increase the proliferation of granulosa cells via PI3K/AKT pathways. These functions are regulated specifically by growth hormones produced by AT-MSCs in response to the surrounding environment, including HGF, TGF-β, IGF-1, and EGF. d AT-MSCs are also good candidates for skin healing and regeneration as their growth factors strongly support neovascularization and angiogenesis by reducing PLL4, increase anti-apoptosis via the activation of PI3K/AKT pathways, regulate inflammation by downregulating NADPH oxidase isoform 1, and increase immunoregulation through the inhibition of NF-κB activation. The figure was created with BioRender.com

COPD is characterized by an increase in hyperinflammatory reactions in the lung, compromising lung function and increasing the development of lung fibrosis. The mechanism by which UC-MSCs contribute to the response to COPD is inflammatory regulation (Fig. ​ (Fig.4b). 4b ). The administration of UC-MSCs prevented the infiltration of inflammatory cells in peribronchiolar, perivascular, and alveolar septa and switched macrophage polarization to M2. 396 A significant reduction in proinflammatory cytokines, including IL-1β, TNF-α, and IL-8, was also observed following UC-MSC administration. 224 MSCs, including UC-MSCs, have been reported to trigger the production of secretory leukocyte protease inhibitors in epithelial cells through the secretion of HGF and epidermal growth factor (EGF), which is believed to have beneficial effects on COPD. 397 , 398 In addition to their inflammatory regulation ability, UC-MSCs exhibit antimicrobial effects through the inhibition of bacterial growth and the alleviation of antibiotic resistance during Pseudomonas aeruginosa infection. 399 The combination of the regulation of the host immune response and the antimicrobial effects of UC-MSCs may be relevant for the prevention and treatment of COPD exacerbations, as inflammation and bacterial infections are important risk factors that significantly contribute to the morbidity and mortality of patients with COPD. In terms of regenerative functions, UC-MSCs were reported to be able to differentiate into type 2 alveolar epithelial cells in vitro and alleviate the development of pulmonary fibrosis via β-catenin-regulated cell apoptosis. 400 Furthermore, UC-MSCs enhanced alveolar epithelial cell migration and proliferation by increasing matrix metalloproteinase-2 levels and reduced their endogenous inhibitors, tissue inhibitors of matrix metalloproteinases, providing a potential mechanism underlying their anti-pulmonary-fibrosis effects. 401 , 402

In ARDS, especially that associated with COVID-19, the proinflammatory state is initiated by increases in plasma concentrations of proinflammatory cytokines, such as IL-1 beta, IL-7, IL-8, IL-9, IL-10, bFGF, granulocyte colony-stimulating factor (G-CSF), GM-CSF, IFN-γ, and TNF-α. The significant increases in the concentrations of these cytokines in patient plasma suggest the development of a cytokine storm, which is a leading cause of COVID-induced mortality. In addition to the immunomodulatory functions regulated via cell-to-cell interactions between UC-MSCs and immune cells, such as macrophages, monocytes, and T cells, UC-MSCs exert their functions via paracrine effects through the secretion of growth factors, cytokines, and exosomes (Fig. ​ (Fig.4c). 4c ). The most relevant immunomodulatory function of UC-MSCs is considered to be their inhibition of effector T cells via the induction of T-cell apoptosis and cell cycle arrest by the production of indoleamine 2,3- dioxygenase (IDO), prostaglandin E2 (PGE-2), and TGF-β. Elevated levels of PGE-2 in patients with COVID-19 are reported to be a crucial factor in the initiation of inflammatory regulation by UC-MSCs post administration and prevent the development of cytokine storms by direct inhibition of T- and B lymphocytes. 403 UC-MSCs exert these inhibitory activities through a PGE-2-dependent mechanism. 404 It was reported that UC-MSCs confer the ability to secrete tolerogenic mediators, including TGF-β1, PGE-2, nitric oxide (NO), and TNF-α, which are directly involved in their immunoregulatory mechanism. The secretion of NO from UC-MSCs is reported to be associated with the desensitization of T cells via the IFN-inducible nitric oxide synthase (iNOS) pathways and to stimulate the migration of T cells in close proximity to MSCs that subsequently suppress T-cell sensitivities via NO. 405 Lung infection with viruses usually leads to impairments in alveolar fluid clearance and protein permeability. The administration of UC-MSCs enhances alveolar protection and restores fluid clearance in patients with COVID-19. UC-MSCs secrete growth factors associated with angiogenesis and the regeneration of pulmonary blood vessels and micronetworks, including angiotensin-1, VEGF, and HGF, which also reduce oxidative stress and prevent fibrosis formation in the lungs. These trophic factors have been identified as key players in the modulation of the microenvironment and promote pulmonary repair. Additionally, UC-MSCs are more effective than BM-MSCs in the restoration of impaired alveolar fluid clearance and the permeability of airways in vitro, supporting the use of UC-MSCs in the treatment of patients with pulmonary pneumonia. 406 In the context of pulmonary regeneration, UC-MSCs were shown to inhibit apoptosis and fibrosis in pulmonary tissue by activating the PI3K/AKT/mTOR pathways via the secretion of HGF, which also acts as an inhibitory stimulus that blocks alveolar epithelial-to-mesenchymal transition. 407 , 408 Moreover, UC-MSCs can reverse the process of fibrosis via enhanced expression of macrophage matrix-metallopeptidase-9 for collagen degradation and facilitate alveolar regeneration via Toll-like receptor-4 signaling pathways. 409 UC-MSCs were shown to communicate with CD4+ T cells through HGF induction not only to inhibit their differentiation into Th17 cells, reducing the secretion of IL-17 and IL-22 but also to switch their differentiation into regulatory T cells. 410 , 411 In addition, UC-MSCs conferred the ability to facilitate the number of M2 macrophages and reduce M1 cells via the control of the macrophage polarization process. 412

There are several potential mechanisms of UC-MSCs in the treatment of patients with pulmonary diseases and pneumonia, including the regulation of immune cell function, immunomodulation, the enhancement of alveolar fluid clearance and protein permeability, the modulation of endoplasmic reticulum stress, and the attenuation of pulmonary fibrosis. Hence, based on these discussions, UC-MSCs are recommended as suitable candidates for the treatment of pulmonary disease both in pediatric and adult patients.

Proposed mechanism of AT-MSCs in the treatment of endocrinological diseases, reproductive disorders, and skin burns

Human AT was first viewed as a passive reservoir for energy storage and later as a major site for sex hormone metabolism, the production of endocrine factors (such as adipsin and leptin), and a secretion source of bioactive peptides known as adipokines. 413 It is now clear that AT functions as a complex and highly active metabolic and endocrine organ, orchestrating numerous different biological features 414 (Fig. ​ (Fig.5a). 5a ). In addition to adipocytes, AT contains hematopoietic-derived progenitor cells, connective tissue, nerve tissue, stromal cells, endothelial cells, MSCs, and pericytes. AT-MSCs and pericytes mobilize from their perivascular locations to aid in healing and tissue regeneration throughout the body. As AT is involved directly in energy storage and metabolism, AT-MSCs are also mediated and regulated by growth factors related to these pathways. In particular, interleukin-6 (IL-6), IL-33, and leptin regulate the maintenance of metabolic activities by increasing insulin sensitivity and preserving homeostasis related to AT. Nevertheless, in the development of obesity and diabetes, omental and subcutaneous AT maintains a low-grade state of inflammation, resulting in the impairment of glucose metabolism and potentially contributing to the development of insulin resistance. 415 In normal AT, direct regulation of Pre-B-cell leukemia homeobox (Pbx)-regulating protein-1 (PREP1) by leptin and thyroid growth factor-beta 1 (TGF-β1) in AT-MSCs and mature adipocytes is involved in the protective function and maintenance of AT homeostasis. However, under diabetic conditions, the balance between the expression of leptin and the secretion of TGF-β1 is compromised, resulting in the malfunction of AT-MSC metabolic activity and the proliferation, differentiation, and maturation of adipocytes. Therefore, the use of autologous AT-MSCs in the treatment of diabetic conditions is not a suitable option, as the functions of AT-MSCs are directly altered by diabetic conditions, which reduces their effectiveness in cell-based therapy (Fig. ​ (Fig.5b 5b ).

Umbilical cord-derived mesenchymal stem cells (UC-MSCs) are good candidates for the treatment of pulmonary diseases. a Lung immaturity and fibrosis are the major problems of patients with bronchopulmonary dysplasia and lead to increased levels of SDF-1, the development of fibrosis, the induction of the inflammatory response, and the impairment of alveolarization. UC-MSCs are attracted to the damaged lung via the chemoattractant SDF-1, which is constantly released from the immature lung via SDF-1 and CXCR4 communication. Moreover, UC-MSCs reduce the level of proinflammatory cytokines (TGF-β, INF-γ, macrophage MIF, and TNF-α) via a cell-to-cell contact mechanism. The ability of UC-MSCs to produce and secrete VEGF also involves in the regeneration of the immature lung through enhanced angiogenesis. b Upon an exacerbation of chronic obstructive pulmonary disease (COPD), UC-MSCs respond to the surrounding stimuli by reducing IL-8 and TNF-α levels, resulting in the inhibition of the inflammatory response but an increase in the secretion of growth factors participating in the protection of alveoli, fluid clearance and reduced oxidative stress and lung fibrosis, including HGF, TGF-β, IGF-1, and exosomes. c In a similar manner, UC-MSCs prevent the formation of cytokine storms in coronavirus disease 2019 (COVID-19) by inhibiting CD34+ T-cell differentiation into Th17 cells and enhancing the number of regulatory T cells. Moreover, UC-MSCs also have antibacterial activity by secreting LL-3717 and lipocalin. Figure was created with BioRender.com

Preclinical studies and clinical trials have revealed the therapeutic effects of MSCs, in general, and AT-MSCs, in particular, in the management of POF, with relatively high efficacy and enhanced regeneration of the ovaries. Understanding the molecular and cellular mechanisms underlying these effects is the first step in the development of suitable MSC-based therapies for POF. One of the mechanisms by which MSCs exert their therapeutic effects is their ability to migrate to sites of injury, a process known as “homing”. Studies have shown that MSCs from different sources have the ability to migrate to different compartments of the injured ovary. For example, BM-MSCs administered through IV routes migrated mostly to the ovarian hilum and medulla, 416 whereas a significant number of UC-MSCs were found in the medulla. 417 Interestingly, AT-MSCs were found to be engrafted in the theca layers of the ovary but not in the follicles, where they acted as supportive cells to promote follicular growth and the regeneration of thecal layers. 418 The structure and function of the thecal layer have a great impact on fertility, which has been reviewed elsewhere. 419 In brief, the thecal layer consists of two distinct parts, the theca interna, which contains endocrine cells, and the theca externa, which is an outer fibrous layer. The thecal layer contains not only endocrine-derived cells but also vascular- and immune-derived cells, whose functions are to maintain the structural integrity of the follicles, transport nutrients to the inner compartment of the ovary and produce key reproductive hormones such as androgens (testosterone and dihydrotestosterone) and growth factors (morphogenic proteins, e.g., BMPs and TGF-β). 420 As AT-MSCs originate from an endocrine organ, their ability to sense signals and migrate to the thecal layer is anticipated. Additionally, secretome analysis of AT-MSCs showed a wide range of growth factors, including HGF, TBG-β, VEGF, insulin-like growth factor-1 (IGF-1), and EGF, 421 that are directly involved in the restoration of the structure and function of damaged ovaries by stimulating cell proliferation and reducing the aging process of oocytes via the activation of the SIRT1/FOXO1 pathway, a key regulator of vascular endothelial homeostasis. 422 , 423 In POF pathology, autophagy and its correlated oxidative stress contribute to the development of POF throughout a patient’s life. Recently, AT-MSCs were shown to be able to improve the structure and function of mouse ovaries by reducing oxidative stress and inflammation, providing essential data supporting the mechanism of AT-MSCs in the treatment of POF. 424 Several studies have illustrated that AT-MSCs secrete biologically active EVs that regulate the proliferation of ovarian granulosa cells via the PI3K/AKT pathway, resulting in the enhancement of ovarian function. 425 Direct regulation of ovarian cell proliferation modulates the state of these cells, which in turn restores the ovarian reserve. 426 Other mechanisms supporting the effectiveness of MSCs have been carefully reviewed, confirming the therapeutic potential of MSCs derived from different sources 426 (Fig. ​ (Fig.5c 5c ).

In the last decade, the number of clinical trials using AT-MSCs in the treatment of chronic skin wounds and skin regeneration has exponentially increased, with data supporting the enhancement of the skin healing processes, the reduction of scar formation, and improvements in skin structure and quality. Several mechanisms are directly linked to the origin of AT-MSCs, including differentiation ability, neovascularization, anti-apoptosis, and immunological regulation. AT is a connective and supportive tissue positioned just beneath the skin layers. AT-MSCs have a strong ability to differentiate into adipocytes, endothelial cells, 427 epithelial cells 428 and muscle cells. 429 The adipogenic differentiation of AT-MSCs is one of the three mesoderm lineages that defines MSC features, and AT-MSCs are likely to be the best MSC type harboring this ability compared to BM- and UC-MSCs. Recent reports detailed that AT-MSCs accelerated diabetic wound tissue closure through the recruitment and differentiation of endothelial cell progenitor cells into endothelial cells mediated by the VEGF-PLCγ-ERK1/ERK2 pathway. 430 Upon injury, the skin must be healed as quickly as possible to prevent inflammation and excessive blood loss. The reparation process occurs through distinct overlapping phases and involves various cell types and processes, including endothelial cells, keratinocyte proliferation, stem cell differentiation, and the restoration of skin homeostasis. 431 Hence, the differentiation ability of AT-MSCs plays a critical role in their therapeutic effect on skin wound regeneration and healing processes. AT-MSCs accelerate wound healing via the production of exosomes that serve as paracrine factors. It was reported that AT-MSCs responded to skin wound injury stimuli by increasing their expression of the lncRNA H19 exosome, which upregulated SOX9 expression via miR-19b, resulting in the acceleration of human skin fibroblast proliferation, migration, and invasion. 432 In addition, the engraftment of AT-MSCs supported wound bed blood flow and epithelialization processes. 433 Anti-apoptosis plays a critical role in AT-MSC-based therapy, as without a microvascular supply network established within 4 days post injury, adipocytes undergo apoptosis and degenerate. Exogenous sources of AT-MSCs mediate anti-apoptosis via IGF-1 and exosome secretion by triggering the activation of PI3K signaling pathways. 434 Another mechanism supporting the therapeutic potential of AT-MSCs is their anti-inflammatory function, which results in the reduction of proinflammatory factors, such as tumor necrosis factor (TNF) and interferon-γ (IFN-γ), and increases the production of the anti-inflammatory factors IL-10 and IL-4. Exosomes from AT-MSCs in response to a wound environment were found to contain high levels of Nrf2, which downregulated wound NADPH oxidase isoform 1 (NOX1), NADPH oxidase isoform 4 (NOX4), IL-1β, IL-6, and TNF-α expression. The anti-inflammatory functions of AT-MSCs are also regulated by their immunomodulatory ability, partially through the inhibition of NF-κB activation in T cells via the PD-L1/PD-1 and Gal-9/TIM-3 pathways, providing a novel target for the acceleration of wound healing 435 (Fig. ​ (Fig.5d 5d ).

Therefore, as an endocrine organ in the human body, AT and its derivative stem cells, including AT-MSCs, have shown great potential in the treatment of reproductive disorders and skin diseases. Their potential is supported by mechanisms that are directly related to the nature of AT-MSCs in the maintenance of tissue homeostasis, angiogenesis, anti-apoptosis, and the regulation of inflammatory responses.

The current challenges for MSC-based therapies

Over the past decades, MSC-based research and therapy have made tremendous advancements due to their advantages, including immune evasion, diverse tissue sources for harvesting, ease of isolation, rapid expansion, and cryopreservation as “off-the-shelf” products. However, several important challenges have to be addressed to further enhance the safety profile and efficacy of MSC-based therapy. In our opinion, the most important challenge of MSC-based therapy is the fate of these cells post administration, especially the long-term survival of allogeneic cells in the treatment of certain diseases. Although reported data confirm that the majority of MSCs are trapped in the lung and rapidly removed from the circulation, caution has been raised related to the occurrence of embolism events post infusion, which was proven to be related to MSC-induced innate immune attack (called instant blood-mediated inflammatory reaction). 436 Another related challenge is the homing ability of infused cells, as successful homing at targeted tissue might result in long-term benefits to patients. Other concerns related to MSC-based therapy are the number of dead cells infused into the patients. An interesting study reported that dead MSCs alone still exerted the same immunomodulatory property as live MSCs by releasing phosphatidylserine. 437 This is an interesting observation, as there is always a certain number of dead cells present in the cell-based product, and concerns are always raised related to their effects on the patient’s health. Finally, the hypothesis presented in this review is also a great challenge of the field, which has been proposed for future studies to answer the question: “What is the impact of MSC sources on their downstream application?”. Tables ​ Tables5 5 and ​ and6 6 illustrate the comparative studies that were conducted in preclinical and clinical settings to address the MSC source challenge. Other challenges of MSC-based therapies have been discussed in several reviews and systematic studies, 135 , 185 , 438 , 439 which are highly recommended.

Comparative analysis of the effectiveness of MSC sources in a preclinical setting

Clinical trials comparing the efficacy of MSCs derived from different sources in the treatment of pulmonary diseases and cardiovascular conditions

Limitations of the current hypothesis

The proposed hypothesis presented in this review was made based on (1) the calculated number of recovered patients from published clinical trials; (2) the empirical experience of the authors in the treatment of brain-related diseases, 440 pulmonary disorders, 215 and endocrinological conditions; 271 , 441 and (3) the proposed mechanisms by which each type of MSC exhibits its best potential for downstream applications. The authors understand that the approach that we used has a certain level of research bias, as a comprehensive meta-analysis is needed to first confirm the correlation between the origins of MSCs and their downstream clinical outcomes before a complete hypothesis can be made. However, to date, a limited number of clinical trials have been conducted to directly compare the efficacy of MSCs from different sources in treating the same disease, which in turn dampened our analysis to prove this hypothesis. In addition, MSC-based therapy is still in its early stages, as controversy and arguments are still present in the field, including (1) the name of MSCs (medicinal signaling cells vs. MSCs or mesenchymal stromal cells), 442 , 443 (2) the existence of “magic cells” (one cell type for the treatment of all diseases), 444 , 445 (3) the conflicting results from large-scale clinical trials, 135 and (4) the dangerous issues of unauthorized, unproven stem cell therapies and clinics. 446 , 447 Therefore, our hypothesis is proposed at this time to encourage active researchers and clinicians to either prove or disprove it so that future research can strengthen the uses of MSC-based therapies with solid mechanistic study results and clarify results for “one cell type for the treatment of all diseases”.

Another limitation is the knowledge coverage in the field of MSC-based regenerative medicine, as discussed in this study. First, the abovementioned diseases were narrowed to four major disease categories for which MSC-based therapy is widely applied, including neuronal, pulmonary, cardiovascular, and endocrinological conditions. In fact, other diseases also receive great benefits from MSC therapy, including liver cirrhosis, 448 bone regeneration, 360 plastic surgery, 449 autoimmune disease, 450 etc., which are not fully discussed in this review and included in our hypothesis. Recently, the secretome profile of MSCs and its potential application in clinical settings have emerged as a new player in the field, with a recently published comprehensive review including MSC-derived exosomes. 451 , 452 To date, the therapeutic potential of MSCs is believed to be strongly influenced by their secretomes, including growth factors, cytokines, chemokines, and exosomes. 453 However, this body of knowledge is also not fully included in our discussion, as this review focuses on the function and potency of MSCs as a whole with considerations derived from published clinical data. Therefore, the authors believe in and support the future applications of the secreted components derived from MSCs, including exosomes, in the treatment of human diseases. In fact, this potential approach could elevate the uses of MSCs to the next level, where the sources of MSCs could be neglected with advancements in the development of protocols that allow strict control of the secretome profiles of MSCs under specific conditions. 454 – 456 Finally, strategies that could potentially enhance the therapeutic outcomes of MSC-based therapy, such as the “priming” process, are not discussed in this review. The idea of “priming” MSCs is based on the nature of MSCs, which is similar to the immune cells, 457 that MSCs have proven to be able to “remember” the stimulus from the surrounding environment. 458 , 459 Thus, activating or priming MSCs using certain conditions, such as hypoxia, matrix mechanics, 3D environment, hormones, or inflammatory cytokines, could trigger the memory mechanism of the MSCs in vitro so that these cells are ready to function towards specific therapeutic activities without the need for in vivo activation. 3 , 460

From a cellular and molecular perspective and from our own experience in a clinical trial setting, AD-, BM- and UC-MSCs exhibit different functional activities and treatment effectiveness across a wide range of human diseases. In this paper, we have provided up-to-date data from the most recently published clinical trials conducted in neuronal diseases, endocrine and reproductive disorders, skin regeneration, pulmonary dysplasia, and cardiovascular diseases. The implications of the results and discussions presented in this review and in a very large body of comprehensive and excellent reviews as well as systematic analyses in the literature provide a different aspect and perspective on the use of MSCs from different sources in the treatment of human diseases. We strongly believe that the field of regenerative medicine and MSC-based therapy will benefit from active discussion, which in turn will significantly advance our knowledge of MSCs. Based on the proposed mechanisms presented in this review, we suggest several key mechanistic issues and questions that need to be addressed in the future:

• The confirmation and demonstration of the mechanism of action prove that tissue origin plays a significant role in the downstream applications of the originated MSCs.
• Is it required that MSCs derived from particular cell sources need to have certain functionalities that are unique to or superior in the original tissue sources?
• As mechanisms may rely on the secretion of factors from MSCs, it is important to identify the specific stimuli from the wound environments to understand how MSCs from different sources can exhibit similar functions in the same disease and whether or not MSCs derived from a particular source have stronger effects than their counterparts derived from other tissue sources.
• Should we create “universal” MSCs that could be functionally equal in the treatment of all diseases regardless of their origin by modeling their genetic materials?
• Can new sources of MSCs from either perinatal or adult tissues better stimulate the innate mechanisms of specific cell types in our body, providing a better tool for MSC-based treatment?
• A potential ‘priming’ protocol that allows priming, activating, and switching the potency of MSCs from one source to another with a more appropriate clinical phenotype to treat certain diseases. This idea is potentially relevant to our suggestion that each MSC type could be more beneficial in downstream applications, and the development of such a “priming” protocol would allow us to expand the bioavailability of specific MSC types.

From our clinical perspective, the underlying proposal in our review is to no longer use MSCs for applications while disregarding their sources but rather to match the MSC tissue source to the application, shifting from one cell type for the treatment of all diseases to cell source-specific disease treatments. Whether the application of MSCs from different sources still shows their effectiveness to a certain extent in the treatment of diseases or not, the transplantation of MSCs derived from different sources for each particular disease needs to be further investigated, and protocols need to be established via multicentre, randomized, placebo-controlled phase II and III clinical trials (Fig. ​ (Fig.6 6 ).

The tissue sources of mesenchymal stem cells (MSCs) contribute greatly to their therapeutic potential, as all MSC types share safety profiles and overlapping efficacy. Although a large body of data and their review and systematic analysis indicated the shared safety and potential efficacy of MSCs derived from different tissue sources, targeted therapies considering MSC origin as an important factor are imperative to enhance the downstream therapeutic effects of MSCs. We suggest that bone marrow-derived MSCs (BM-MSCs) are good candidates for the treatment of brain and spinal cord injury, adipose tissue-derived MSCs (AT-MSCs) are suitable for the treatment of reproductive disorders and skin regeneration, and umbilical cord-derived MSCs (UC-MSCs) could be alternatives for the treatment of pulmonary diseases and acute respiratory distress syndrome (ARDS). Figure was created with BioRender.com

Acknowledgements

The authors would like to thank the Vingroup Scientific Research and Clinical Application Fund (grant number: PRO. 19.47) for supporting this work. All figures were created with Biorender.com. This work is supported by the Vingroup Scientific Research and Clinical Application Fund (Grant number: PRO.19.47).

Author contributions

D.M.H.: conception and design, manuscript writing, administrative support, data analysis and interpretation, and final approval of the manuscript. P.T.P.: manuscript writing (BM- and UC-MSC sections) and data analysis and interpretation. T.Q.B.: manuscript writing (BM- and UC-MSC sections) and data analysis and interpretation. A.T.L.N.: manuscript writing (UC-MSC section), figure presentation, and data analysis and interpretation. Q.T.N., T.T.K.P., G.H.N., P.T.T.L., and V.T.H.: manuscript writing and data analysis and interpretation. N.R.F. and M.H.: manuscript writing and editing and data analysis and interpretation. L.T.N.: manuscript writing, administrative support, and final approval of the manuscript. All authors have read and approved the article.

Data availability

Competing interests.

The authors declare no competing interests.

• Open access
• Published: 08 May 2022

Mesenchymal stromal cells (MSCs) and their exosome in acute liver failure (ALF): a comprehensive review

• Samin Shokravi 1 ,
• Vitaliy Borisov 2 ,
• Burhan Abdullah Zaman 3 ,
• Firoozeh Niazvand 4 ,
• Raheleh Hazrati 5 ,
• Meysam Mohammadi Khah 6 ,
• Lakshmi Thangavelu 7 ,
• Sima Marzban 1 ,
• Armin Sohrabi 8 , 9 &
• Amir Zamani 10  

Stem Cell Research & Therapy volume  13 , Article number:  192 ( 2022 ) Cite this article

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Recently, mesenchymal stromal cells (MSCs) and their derivative exosome have become a promising approach in the context of liver diseases therapy, in particular, acute liver failure (ALF). In addition to their differentiation into hepatocytes in vivo, which is partially involved in liver regeneration, MSCs support liver regeneration as a result of their appreciated competencies, such as antiapoptotic, immunomodulatory, antifibrotic, and also antioxidant attributes. Further, MSCs-secreted molecules inspire hepatocyte proliferation in vivo, facilitating damaged tissue recovery in ALF. Given these properties, various MSCs-based approaches have evolved and resulted in encouraging outcomes in ALF animal models and also displayed safety and also modest efficacy in human studies, providing a new avenue for ALF therapy. Irrespective of MSCs-derived exosome, MSCs-based strategies in ALF include administration of native MSCs, genetically modified MSCs, pretreated MSCs, MSCs delivery using biomaterials, and also MSCs in combination with and other therapeutic molecules or modalities. Herein, we will deliver an overview regarding the therapeutic effects of the MSCs and their exosomes in ALF. As well, we will discuss recent progress in preclinical and clinical studies and current challenges in MSCs-based therapies in ALF, with a special focus on in vivo reports.

Introduction

Acute liver failure (ALF) is characterized by the occurrence of coagulopathy (international normalized ratio [INR] > 1.5) and any level of encephalopathy emerging 24 weeks following the occurrence of the first symptoms in patients who have no history of previous liver disease [ 1 ]. The timing and the level of clinical presentation can be classified into three types: hyperacute, acute, and subacute [ 2 ]. Hyperacute and acute types involve fulminant hepatic failure, while the subacute type is also named subfulminant [ 3 ]. Interestingly, the mortality rate among the patients whose hepatic encephalopathy appears 8 weeks after the onset of symptoms (fulminant hepatic failure) is lower than the patients with a more gradually evolving course [ 4 ]. Multiorgan failure (MOF) has proved to be the main cause of death (> 50%) from ALF, while intracranial hypertension (ICH) and infection are the other main causes of mortality in this patient population [ 5 ].

During last two decades, a diversity of stem cells, such as pluripotent stem cells (PSCs), mesenchymal stromal cells (MSCs), hepatic progenitor cells (HPCs), and hematopoietic stem cells (HSCs), has been used to treat liver diseases [ 6 , 7 , 8 ]. However, MSCs are the most common type used in research, since they pose no ethical challenges and can be obtained easily [ 9 , 10 ]. Results show that MSCs have the capability of differentiating more than once; moreover, they can self-renew. They can differentiate into an array of cell lineages, including hepatocyte-like cells (HLCs) [ 11 ]. MSCs are also characterized by other properties, such as anti-inflammatory, antiapoptosis, antifibrotic, antioxidant, blood vessel formation, improvement of tissue repair, and growth factor secretion [ 12 , 13 ]. Despite many preclinical and clinical investigations on MSCs used in treating ALF, it is still unknown what mechanism contributes to the therapeutic effect of MSCs. Besides, MSCs-exosomes have caught the attention of many researchers as a new cell-free method regarding the regeneration of the liver [ 14 , 15 ]. They have dissipated the worries concerning the direct application of MSC (e.g., immunogenicity and tumor formation [ 16 ]. Such exosomes encompass high frequencies of cytoplasmic and membrane proteins, including enzymes, transcription factors, lipids, ECM proteins. They also include nucleic acids, such as mitochondrial DNA (mtDNA), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), messenger RNA (mRNA), and microRNA (miRNA) [ 17 ]. The size of exosomes varies from 30 to 150 nm, and they can be transferred to other cells to do their functions. As a highly controlled process, the generation of exosome from the other organisms similar to themselves is comprised of three main steps: (1) formation of endocytic vesicles by the folding of the exterior area of the plasma membrane, (2) formation of multivesicular bodies (MVBs) by inward budding of the endosomal membrane, and (3) incorporation of established MVBs with the plasma membrane and secretion of the vesicular contents, called exosomes [ 14 , 18 ]. Exosomes elicit antioxidant effects and motivate target cells to trigger downstream signals. Moreover, they convey genetic material to target cells, leading to the suppression of inflammation and apoptosis. [ 19 , 20 ].

This review aims to give an overview of the present knowledge to elucidate mechanisms used by MSCs to underlie liver restoration in ALF. Another aim is to present a discussion of new developments in preclinical and clinical investigations on MSCs therapy in liver-associated diseases, with a particular focus on ALF.

Pathophysiology of ALF

Acetaminophen (APAP) has proved to be the main cause of ALF [ 21 ]. The following people are highly likely to experience ALF stimulated by APAP: alcoholic people who use APAP; people who suffer from malnutrition; or people who take medications that are believed to induce CYP450 enzymes (e.g., phenytoin, carbamazepine, or rifampin). Results of a study on 308 patients with severe liver disorder in the USA revealed APAP as the main cause of ALF in 40% of patients [ 22 ]. The other causes detected were as follows in the increasing order of prevalence:

Malignancy (1%)

Budd-Chiari Syndrome (2%)

Pregnancy (2%)

Wilson disease (3%)

Hepatitis A virus infection (4%)

Autoimmune hepatitis (4%)

Ischemic hepatitis (6%),

Hepatitis B virus infection (6%)

Idiosyncratic drug-induced liver injury (13%)

The causes of 17 percent of cases were not known [ 4 ].

Based on results, it is possible to categorize the ALF pathophysiology into two groups: pathophysiologies of liver problems involving a specific cause and pathophysiology concerning the appearance of secondary multiorgan failure (MOF) [ 23 ]. With regard to the pathophysiology of liver disorders, the results show that APAP toxicity is the main cause [ 24 ]. Secondary MOF often derives from the primary extensive pro-inflammatory effect, which leads to a pervasive inflammatory effect syndrome. Then, a deregulated anti-inflammatory response ensues [ 25 , 26 ].

It is not clear what mechanism causes the ongoing death of tissue when there is no ongoing injury. Oxidative stress results in the formation of reactive oxygen species (ROS). This, in turn, activates c-Jun N-terminal kinase (JNK) through a series of events [ 27 ]. Such activation may support disruption of normal mitochondrial function , which inspires more hepatocyte necrosis and damage associated molecular patterns (DAMPs) [ 28 , 29 ]. DAMPs bring about the activation of hepatic macrophages, resulting in the formation of the inflammasome [ 30 , 31 ]. Concisely, as complexes characterized by multiple proteins, inflammasomes receive the intracellular danger signals through NOD-like receptors (NLRs) [ 32 ]. The inflammasome effectively regulates the inflammatory response by eliciting a response to low-threshold signals. Toll-like receptors (TLRs) induction by DAMPs leads to the inflammasome activation, supporting the subsequent activation of caspase-1 and IL-1β secretion [ 33 , 34 ]. Researchers have identified the characteristics of the NLR family pyrin domain containing 3 (NLRP3) inflammasome belonging to the inflammasome family. NLRP3 inflammasome has three potential activation pathways: (1) ATP signal which occurs outside a cell, leading to potassium efflux and pannexin recruitment; (2) incorporation of crystalized cholesterol, uric acid or amyloid with lysosomal dysfunction after the ingestion and elimination of these particles; and (3) activation by reactive oxygen species (ROS) [ 33 , 35 , 36 ]. Investigations have examined the activation of inflammasome in APAP-induced ALF by a special focus on the contribution of the inflammasome to acute liver disorder [ 37 ]. It appears that DAMPs are released from necrotic hepatocytes and sinusoidal endothelial cells, leading to the activation of the inflammasome in the manner mentioned above.

The rationality of MSCs therapy in ALF

MSCs migration to damage tissue by interaction with several receptors and molecules, and thereby inducing liver recovery by various mechanisms has been proved (Fig.  1 ). Although the mechanisms of MSCs transplantation are still not entirely understood, a growing body of proof has indicated that their immunomodulation, differentiation, and antifibrotic capabilities play central roles in liver repair. Among them, anti-inflammatory potential of MSCs play most critical role. Although there is no clear evidence indicating the MSCs in vivo differentiation into hepatoid cells post-transplantation, MSCs can be differentiated into hepatocyte-like cells (HLCs) in vitro and then be infused. Of course, this process is time-consuming process with insufficient established HLCs, thereby hindering its therapeutic utility in clinic. However, there is some evidence indicating that replacing fetal bovine serum (FBS) with polyvinyl alcohol (PVA) might lead to improved differentiation ability [ 38 ]. In vivo, as only a small number of intravenously injected cells reach the liver, MSCs-mediated favored effects mainly depend on the secreted molecules rather than their direct effects or differentiation into hepatocytes post-transplantation [ 39 ].

Underlying mechanism complicated in mesenchymal stromal cells (MSCs) migration to damaged liver tissue. The connections between CXCR4 and SDF-1ɑ, c-Met and HGF, and finally VLA-4 and VCAM-1 underlie cell to cell interaction between endothelial cells (ECs) and MSCs, which, in turn, facilitate MSCs migration to damaged liver tissue. Then MSCs secrete anti-inflammatory molecules, such as PGE2, IDO, TGF-β, IL-10, and NO to down-regulate inflammation. These molecules prompt the change of inflammatory to proliferating phase largely defined by the secretion of PDGF and VEGF, sustaining hepatocyte formation and proliferation

MSC anti-inflammatory properties

The hepatocyte loss is the first symptom and mechanism contributing to acute liver damage. It is still unclear what causes ongoing necrosis when there is no injury. ROS are produced in response to oxidative stress. This, in turn, activates c-Jun N-terminal kinase (JNK) through a series of events, resulting in mitochondrial dysfunctions. These events lead to a higher level of hepatocyte necrosis, as well as the expansion of DAMPs [ 40 ]. DAMPs stimulate the activation of hepatic macrophages and the formation of the inflammasome [ 41 ]. In the next stage, the release of pro-inflammatory cytokines eases the recruitment of a larger number of immune cells to the inflammation area, and so advances hepatocyte cell necrosis.

The majority of past investigations have indicated that MSCs play a therapeutic role in the treatment of liver dysfunction by releasing trophic factors and the factors modulating the immune system [ 42 ]. Although the role of MSCs in modulating the immune system is unclear, they might control the immune cells through the secretion of soluble factors and the contacts between cells. The regulation of adaptive and innate immune responses by MSCs is exerted by inhibiting T cells and dendritic cells (DCs), which leads to a reduction in the activation and growth of B cells [ 43 , 44 ]. This, in turn, enhances the formation of regulatory T (Treg) cells and prevents the growth and toxicity of natural killer (NK) cells induced by the chemotherapeutic molecules [ 45 ]. Also, transforming growth factor-beta (TGF-β) and interleukin 10 (IL-10) as crucial factors in the regulation of a large number of inflammatory cells [ 46 , 47 ]. Studies revealed a significant increase in the amounts of TGF-β and IL-10 in serum following the injection of UC-MSCs, but a significant decrease in the amounts of IL-6, tumor necrosis factor-alpha (TNF-α), and cytotoxic T lymphocytes (CTLs) was seen in peripheral blood [ 48 ]. This led to the restoration of liver function, as well as a reduction in the development of disease and the level of mortality. Furthermore, transient T cell apoptosis can be induced by BM-MSCs through the Fas ligand (FasL)-dependent pathway [ 49 ]. Then, macrophages are stimulated by apoptotic T cells to form high amounts of TGF-β, resulting in the up-regulation of Treg cells to trigger immune tolerance. MSCs can prevent cytotoxic CTLs and NK cells through the contact between cells and paracrine factors, including indoleamine 2,3-dioxygenase (IDO), TGF-β, and prostaglandin E2 (PGE2) [ 50 , 51 ]. Of course, TGF-β acts as a two-edged sword. It can weaken the immune system and thereby suppress liver inflammation [ 49 ]; on the other hand, it can increase liver fibrosis [ 52 ]. MSCs, in fact, can act as an immunomodulatory agent in reducing the inflammation of the body through up-regulating anti-inflammatory Treg cells and decreasing T helper 1 (Th1) and Th17 cells in ALF [ 53 ]. Moreover, the inflammation after MSC transplantation can be indirectly stimulated by up-regulating M2-type macrophages, leading to the secretion of a variety of anti-inflammatory factors, such as chemokine ligand 1 (CCL-1) and IL-10, up-regulation Th2, and Treg cells [ 54 ]. Also, MSCs play an important role in the reduction of B-cell growth through contact between cells and the secretion of soluble factors [ 55 ]. Finally, MSC transplantation can play an effective role in mitigating liver damage in ALF by decreasing the number and activity of neutrophils in both peripheral blood and the liver.

MSCs differentiate into HLCs

MSCs are characterized by their ability to proliferate and differentiate in vitro. For the first time, Friedenstein et al. procured MSCs in 1968 from the bone marrow (BM) [ 56 , 57 ]. After that, MSCs obtained from multiple sources, making them an excellent supply of multipotent cells for treatment of liver dysfunctions. A variety of methods are used to differentiate MSCs into HLCs [ 58 , 59 ]. Studies show that multiple signals contribute to the regulation of the cells’ behavior in a cooperative manner. Such signals are usually triggered by extracellular matrix (ECM), growth factors, and even juxtacrine signals [ 60 ]. Each one of the organs, as well as the developmental stage, is characterized by a specific regulated timing and distribution pattern of signals. As a result, achieving better results in the case of in vitro cultures requires establishing a type of environment that resembles the local environment. Based on the research previously done, it is possible to differentiate MSCs obtained from various sources into hepatocytes in the case of both mice and humans through the implementation of a variety of protocols and methods in vitro [ 61 , 62 ].

Hepatic differentiation protocol is known to be the most frequently used method for hepatic differentiation, which benefits from Iscove's Modified Dulbecco's Medium (IMDM), as well as cytokine cocktail. Epidermal growth factor (EGF) or fibroblast growth factors (FGF) trigger the MSCs to differentiate into endodermal cells during the early induction stage. EGF prompts the MSCs to proliferate by interfaces with EGF receptor (EGFR) [ 63 ]. Besides, FGF is a member of a bigger family that is comprised of seven polypeptides with similar characteristics [ 64 ]. This family plays an essential role in the primary stage of endodermal patterning [ 65 ]. In particular, FGF-4 and basic FGF (bFGF) are commonly used. Like EGF, FGF influences the growth rate of MSCs [ 66 ].

Generally, the differentiation of cells is stimulated by adding FGF, HGF, nicotinamide (NTA), and also insulin-transferrin-selenium (ITS) into cultures [ 67 ]. As a mesenchymal origin pleiotropic cytokine, hepatocyte growth factor (HGF) contributes to adjustment of growth, differentiation, and chemotactic migration of MSCs [ 68 ]. MSCs’ exposure to HGF for a short time causes the activation of c-Met receptors along with its downstream agents such as extracellular signal-regulated protein kinase (ERK)1/2, p38, mitogen-activated protein kinases (MAPKs), and phosphoinositide 3-kinase (PI3K) /Akt [ 69 , 70 ]. MSCs’ exposure to HGF for a long time will make changes in cytoskeletal arrangement; moreover, it results in the migration of cells and a notable decrease in proliferation. In addition, ITS and NTA promote the growth and survival of primary hepatocytes [ 71 ].

Despite the fact that MSCs can differentiate in culture through induction, the organ-specific microenvironment is the best technique, enabling MSCs differentiation into a certain cell type. The ability to express hepatocyte-specific genes is one of the specific characteristics of hepatic-differentiated cells, which can be affected by microenvironmental features [ 72 ]. Reports display that in the case of humans, the differentiation of the MSCs obtained from umbilical cord (UC) into HLCs occurs more quickly in the fibrotic liver microenvironment [ 73 ].

Other studies also show that the differentiation of MSCs into functional hepatocytes does not occur directly; rather, these cells initially differentiate into biliary epithelial cells (BEC)-like cells, followed by differentiation into HLCs [ 74 ]. However, according to the results of other investigations, MSCs transdifferentiation infrequently occurs after MSC infusion in animal models [ 75 ]. MSCs obtained from menstrual blood, for instance, turned out to prevent hepatic satellite cells (HSCs) activation and resultant liver fibrosis, leading to the improvement of liver function. Yet, very few transplanted MSCs differentiated into functional HLCs in vivo [ 76 ]. These results demonstrate that the therapeutic impact of MSCs is mediated mainly by indirect paracrine signaling.

MSCs antifibrotic properties

Thanks to their antifibrotic and immunosuppressive properties, MSCs play an important role in the treatment of fibrosis [ 77 ]. Also, fibrosis in not a common pathological signs of ALF; long-term liver damage mainly results in fibrosis. MSC transplantation could attenuate liver fibrosis by down-regulation of TGF-β1, Smad2, collagen type I, and smooth muscle alpha-actin (αSMA), reducing liver fibrosis regions in vivo [ 78 ]. Besides, BM-MSCs decreased hepatic collagen distribution by impairing the TGF-β/Smad signaling pathway in a cirrhosis rat models [ 79 ]. MSCs also ameliorated hepatic microvascular dysfunction and portal hypertension, which contribute to complications defining clinical decompensating [ 80 ]. Further, the expression of matrix metalloproteinase (MMP)-2, -9, -13, and -14 can be up-regulated by MSCs [ 81 ], which, in turn, attenuates liver fibrosis through degrading extracellular matrix (ECM) [ 82 ]. MSCs reinforce this effect by the down-regulation of the tissue inhibitors of matrix metalloproteinases (TIMPs). Importantly, there is an association between the balanced levels of MMPs and TIMP and fibrosis resolution [ 83 ]. Moreover, MSCs have both direct and indirect roles in inhibiting the activation and growth of hepatic satellite cells (HSCs) and thereby could inhibit collagen synthesis [ 84 ]. The direct interactional relationship between MSCs and HSCs helps to inhibit HSC proliferation by stimulating G0/G1 cell-cycle arrest. This is done by inhibiting the phosphorylation of ERK1/2 [ 85 ]. On the other hand, MSCs contain substantial levels of milk fat globule-EGF factor 8 (MFGE8). The MFGE8 reduces expression levels of TGF-β1 receptor on HSCs, thus strikingly fences primary human HSCs activation [ 86 ]. In co-culture conditions, MSCs also mainly impair α-smooth muscle actin (α-SMA) expression of HSCs, which is mediated, in part, by the activation of the Notch pathway [ 87 ]. The indirect secretion of some pivotal factors (IL-10, HGF, TGF-β, and TNF-α) by MSCs averts the growth of HSCs and reduces the formation of collagen. In contrast, HGF and NGF enhance the apoptosis of HSCs [ 88 , 89 ].

MSCs antioxidant properties

One of the events deriving from ROS is oxidative stress, which is known as a common driver in creating damage to the liver. Some of these damages include the liver failure, liver fibrosis, liver cirrhosis, viral hepatitis, and also hepatocellular carcinoma (HCC) [ 90 , 91 ]. The results of some investigations have revealed that MSCs play a strong mediatory antioxidant role in different animal models [ 92 , 93 ]. Oxidative liver injury is mostly caused by thioacetamide (TAA) or carbon tetrachloride (CCl4) as the most commonly used toxins. These types of toxins give rise to hepatocyte dysfunction through the peroxidation of lipid and proteins alkylation, nucleic acids, and lipids [ 94 , 95 ], resulting in the inflammatory response, hepatocellular injury, and liver fibrosis. Cell signaling and homeostasis require a low level of physiologic ROS formed by the mitochondrial respiration. MSCs have proved to have the capability of mitigating oxidative stress simulated by CCl4 and TAA in vivo [ 96 , 97 ]. Through enhancing the superoxide dismutase (SOD) expression and antioxidant response elements (AREs) stimulation, MSCs boost antioxidant and cytoprotective performance, leading to a reduction in hepatocyte apoptosis [ 98 , 99 ]. Due to their antioxidant role along with their role in modulating the immune systems, MSCs can be very useful in developing therapies for liver injuries.

The importance of MSCs-exosome as cell-free approach in ALF

Exosome is a main subtype of extracellular vesicles (EVs) with a diameter in the range of 40–150 nm. Exosome are mainly produced by a diversity of human cells, such as stem/stromal cells, immune cells, and tumor cells [ 100 ]. They include several biological components, more importantly, miRNAs, proteins, lipids and mRNAs, as cargo [ 101 , 102 ]. The production and secretion procedure of exosome consists of three chief steps: (1) creation of endocytic vesicles through invagination of the plasma membrane, (2) creation of multivesicular bodies (MVBs) upon endosomal membranes’ inward budding, and (3) incorporation of created MVBs with the plasma membrane and releases of the vesicular contents termed exosomes [ 14 , 18 ]. Then, the contents of exosomes are transferred to the recipient cells, and thereby modify physiological cells [ 15 ]. As a result of their great capabilities to elicit hepatoprotective effect, exosomes are recently been considered as a rational therapeutic option for liver failure, thereby circumventing comprehensions concerning stromal cells’ direct transplantation [ 103 , 104 ]. They are smaller and less complex compared with parent cells, and thereby easier to produce and store. Also, they exhibit no risk of tumor formation. Importantly, exosomes are less immunogenic than their parent cells due to their lower membrane-bound proteins. Recently, UC-MSCs-derived glutathione peroxidase1 (GPX1) enriched exosome showed the capacity to compromise oxidative stress as well as apoptosis in the hepatocyte, stimulating hepatoprotective effect in ALF rodent models [ 105 ]. Also, MSCs-derived exosome potently reduced inflammatory response in ALF animal models by impairment of IL-6-mediated signaling axis [ 106 ] and also down-regulation of NLRP3 pathway [ 107 ]. However, further studies are strongly needed to entirely elucidate how MSCs-derived exosomes exert their hepatoprotective influences in vivo.

MSCs in ALF (animal studies)

Native mscs.

MSCs-based treatments have shown huge potential for regenerating the liver and repairing its injury, which resulted from several liver disorders (Tables 1 , 2 ). In vivo, MSCs can migrate to damaged tissues and constrain the production of pro-inflammatory molecules (e.g., IL-1, IL-6, and TNF-ɑ) and ultimately potentiate liver cells growth. As described, the chief mechanism behind the MSCs-mediated positive effects is their immunoregulatory potential rather than direct differentiation into haptoid cells. These cells efficiently hinder the activation of both innate and adaptive immune system cells, such as neutrophils, macrophages, NK cells, DCs, monocytes, and also lymphocytes. Studies in liver failure animal models revealed that MSCs could transdifferentiate into albumin-expressing HLCs, and also may support normal hepatocytes proliferation in vivo upon fusion with them [ 108 ]. Findings have outlined the important roles of SDF-1/CXCR4 axis to ease MSCs migration to damaged tissue, sustaining liver rescue in ALF [ 108 ]. As well, injection of MSCs-derived hepatocyte into mice with liver failure ameliorated liver function, as evidenced by analysis of serum profile as well as biochemical factors rates [ 109 ]. Notably, the serum levels of TGF-β1 and IL-10 in transplanted animals were more prominent than in control animals [ 109 ]. Other studies displayed that pyroptosis, a unique shape of programmed cell death induced by penetrating inflammatory reaction, was suppressed by MSCs therapy in ALF preclinical model [ 110 ]. Accordingly, MSCs administration caused liver repair in C57BL/6 mice by up-regulation of IL-10 and concomitantly suppression of NLRP3 [ 110 ]. Given that NLRP3 inflammasome elicits liver failure through induction of procaspase-1 and pro-IL-1 β accompanied with the adjustment of downstream CD40-CD40L signaling, its inhibition as elicited by MSCs can enable liver recovery in ALF [ 111 ]. Besides, the study of the soluble factor produced by MSCs and their potent desired impacts in a rat model of ALF revealed that IL-10, which mainly is secreted by MSCs, has a central role in ALF recovery post-transplantation [ 112 ]. It was found that phosphorylated STAT3 diminished upon IL-10 injection and conversely STAT3 suppression abrogated IL-10-induced effects in vivo, reflecting the eminent role of STAT3 signaling in exerting IL-10-induced anti-inflammatory influences [ 112 ]. In addition to the IL-10, MSC-produced PGE2 could constrain apoptosis and simultaneously augment hepatocyte proliferation, thereby decreasing ALF [ 113 ]. In fact, PGE2 stimulated YAP activation and then activated YAP suppressed phosphatase and tensin homolog (PTEN) and consequently up-regulated mammalian target of rapamycin (mTOR), a foremost controller of cell growth. This axis in turn protected versus ALF through increasing hepatocyte proliferation [ 113 ]. Furthermore, there is clear evidence signifying that MSCs could modify phenotype and function of macrophages, adjust DCs either differentiation or maturation, and impede the T cell activities by the production of tumor necrosis factor-alpha-stimulated gene 6 (TSG-6) in ALF models [ 114 ]. TSG-6 mainly averts the inflammatory response as a result of suppressing P38 and JNK signaling axes, providing a suitable milieu for ALF rescue upon MSCs transplantation [ 115 ]. MSCs also can induce their favored influences by heme oxygenase (HO) 1, a rate-limiting enzyme in heme metabolism, which is noted as an effective antioxidative and cytoprotective molecule. Recently, it was proven that MSCs administration gave rise to HO-1 up-regulation, whereas suppressing HO-1 impaired MSCs-induced desired effects and also hepatocyte autophagy [ 116 ]. These favored effects upon MSCs therapy were most probably caused by PI3K/Akt signaling pathway-induced HO-1 up-regulation [ 116 ]. Also, Zhang et al. found that systemic administration of BM-MSCs into the ALF rat model attenuated ALF by up-regulation of the HO-1 expression and subsequent attenuation in neutrophil infiltration and activation [ 117 ]. This event finally reduced hepatocyte apoptosis and also improve their proliferation, culminating liver recovery. Similarly, the pivotal role of neutrophils in ALF pathogenesis has been clarified by other reports [ 118 ]. In the D-galactosamine-induced ALF animal model, the great number of neutrophils aggregated in the liver tissue along with promoted myeloperoxidase (MPO) activity and enhanced alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum levels are mainly detected [ 118 ]. Nonetheless, injection of BM-MSCs brought about functional recovery, which was documented by reduced ALT and AST levels and also improved survival rate in the treatment group compared with the control group (50% vs 12.5%). Notably, the intervention led to a robust decrease in the population of neutrophils in the liver, MPO function, and also the expression of pro-inflammatory factors, including TNF-α, IL-1β, interferon gamma (IFNγ) and CXC chemokine ligands 1/2 (CXCL1/2) [ 118 ]. In addition, in a monkey model of ALF, systemic administration of the MSCs derived from another source, unbiblical cord (UC), reduced hepatic aggregation and maturation of circulating monocytes and their IL-6 releases, resulted in prolonged survival [ 119 ]. UC-MSCs also could induce a reduction in ALF by provoking the endogenous liver regeneration in association with suppression of liver cell apoptosis by up-regulating HGF/c-Met signaling axis [ 120 ] or down-regulation of Notch and STAT1/STAT3 signaling [ 121 ]. The positive influences of MSC therapy on hepatocyte proliferation also may arise from activation of AKT/ glycogen synthase kinase 3 beta (GSK-3β)/β-catenin pathway and enhancement in glucose metabolism leading to improved survival rate in ALF animal model [ 122 ]. Interestingly, intraportal injection of MSCs showed superiority over hepatic intra-arterial, intravenous, and intrahepatic injection in terms of liver recovery rate in swine with ALF. Notably, the liver recovery might be attributable to down-regulation of caspase-3, up-regulation of apoptosis inhibitor survivin as well as activation of AKT and ERK axes [ 123 ]. On the other hand, another study revealed that systemic infusion of MSCs was more effective than the intraperitoneal (IP) injection to support liver recovery because of the more significant increase in expression levels of growth factor vascular endothelium growth factor (VEGF) [ 124 ]. Also, compared with BM-MSC, adipose tissue (AT)-derived MSCs displayed higher therapeutic capacities, as defined by estimation of ALT and AST levels post-transplantation in ALF murine model [ 125 ].

MSCs delivery using biomaterials

Present cell transplantation approaches are hindered via poor post-delivery survival, liver ECM and vasculature deterioration, and also difficulties in fusion into the host tissue [ 126 ]. As a result, scientists are persuaded to deliver MSCs within biomaterial structure to sustain the transplants’ viability and also potentiate MSCs long-standing activation in vivo [ 126 ].

Recent reports noted that BM-MSCs are valued options to co-culture with hepatocytes in poly (lactic acid-glycolic acid) (PLGA) scaffolds, enhancing the hepatocellular activities [ 127 ]. Administration of BM-MSCs and hepatocyte seeded PLGA scaffolds led to the considerably advanced cellular proliferation and conversely supported a striking reduction in ALT, AST, and total bilirubin in ALF preclinical models post-transplantation, ultimately leading to the prolonged survival [ 127 ]. Another study demonstrated that MSCs seeded PLGA scaffolds were survived for 3 weeks, and displayed more evident activities than MSCs injected by intravenous route, which was verified by lower mortality in vivo [ 128 ]. However, there was no significant alteration in hepatic inflammation and necrosis zones between the two applied interventions [ 128 ]. Also, poly L-lactic acid (PLLA) nanofiber scaffold could improve the hepatic differentiation of BM-MSCs [ 129 ]. In vitro, analysis exhibited that expression levels of liver-specific markers, more importantly, albumin and α-fetoprotein, were greater in differentiated cells on the nanofibers compared with differentiated cells in plates. These results deliver the proof of the theory that engineered PLLA scaffold could be an efficient alternative to augment MSCs differentiation into functional HLCs [ 129 ]. Besides, BM- and AT-MSC seeded regenerated silk fibroin (RSF) matrices potently differentiated into HLCs in vitro and also stimulated angiogenesis and restored liver functions in the ALF mice model in vivo [ 130 ].

Combination therapy with MSCs

A diversity of studies recently has focused on combination therapy with MSCs and other molecules or modalities to diminish ALF. Meanwhile, co-administration of MSCs with Icaritin, a well-known ingredient isolated from traditional Chinese medicine, resulted in promising outcomes in vivo [ 131 ]. Indeed, MSCs co-cultured with Icaritin improved survival, reduced serum levels of AST and ALT, and elicited histological variations in liver tissue more potently than MSCs alone. Importantly, the up-regulation of HGF/c-Met by Icaritin was found to be involved in MSCs-triggered antiapoptotic influences on hepatocytes, reflecting the potential of herbal extracts to promote MSC-mediated therapeutic impacts [ 131 ]. The addition of the granulocyte colony-stimulating factor (G-CSF) to UCB-MSCs also improved survival and reduced ROS and pro-inflammatory cytokines expressions in ALF murine model [ 132 ]. Also, intervention engendered a significant reduction in cell apoptosis in liver tissues more evidently than UCB-MSCs alone [ 132 ]. These findings were similar to previous reports displaying that G-CSF therapy alone could significantly attenuate short-term mortality in patients suffering from liver failure mainly by reducing inflammation concomitant with activating PI3K/Akt axis in hepatocytes [ 133 , 134 ]. In another study, thanks to the crucial role of IL-1 in the progress of ALF, Sang et al. evaluated possible synergetic effects of combined use of MSCs with 2 mg/kg interleukin-1 receptor antagonist (IL-1Ra) in vivo [ 135 ]. They found that treatment significantly attenuated liver cell apoptosis, improved their proliferation, and eventually enhanced animal survival. It is supposed that the observed effects were dependent on enhancement in AKT and also a reduction in nuclear factor (NF)-κB expression, potentiating liver cell proliferation [ 135 ]. Similarly, co-administration of MSCs plus IL-1Ra chitosan nanoparticles (NPs) was more effective than MSC transplantation alone for treating ALF [ 136 ]. IL-1Ra-loaded NPs administration by ear veins exhibited synergistic impacts with portal vein injection of MSC in a mini swine model of ALF by the hindrance of liver inflammation [ 136 ].

Pretreated MSCs

Current studies have verified that pretreatment with chemical agents, hypoxia, and also cytokine or chemokine in vitro can improve the therapeutic merits of MSCs upon transplantation in vivo [ 137 , 138 ]. Compared to native MSCs, pretreated MSCs largely demonstrate developed hepatogenic differentiation, homing capability, and survival and paracrine impacts.

In 2019, Nie et al. suggested that IL-1β pretreatment could circumvent the MSC's poor migration toward the injured region in ALF murine model [ 139 ]. Correspondingly, IL-1β-MSCs showed superiority over native MSCs respecting the survival time and liver function in vivo. Remarkably, IL-1β-MSCs suppressed liver cell apoptosis and necrosis and also provoked their proliferation. Preferred effects were most probably enticed by improved CXCR4 expression resulting from IL-1β pretreatment and thereby increased migration toward CXCL12 (SDF-1 α) in damage tissue [ 139 ]. Interestingly, pretreatment with injured liver tissue might improve MSCs homing and also their hepatogenic differentiation [ 140 ]. In vivo, transplantation of pretreated MSCs led to an enhancement in albumin, cytokeratin 8, 18, and antiapoptotic protein Bcl-xl levels, whereas supported a reduction in pro-apoptotic protein Bax and caspase-3 levels [ 140 ]. Likewise, short-term, but not long-term, sodium butyrate (NaB) treatment augmented hepatogenic differentiation of BM-MSCs and consequently alleviated liver injury in vivo, according to Li et al. reports [ 141 ].

Genetically modified MSCs

Genetically modified MSCs mainly are used to enhance their colonization rate post-transplantation, leading to ameliorated liver recovery in ALF. Meanwhile, genetically modified MSC to overexpress the CXCR4 gene demonstrated more appropriate migration capability toward SDF-1α and also induce better hepatoprotective impacts in vitro [ 142 ]. In ALF murine model, CXCR4-MSCs efficiently migrated to damaged tissue, and in turn, brought about prolonged survival and restored liver activity more prominent than native MSCs transplantation [ 142 ]. Besides, UCB-MSCs modified to overexpress vascular endothelial growth factor 165 (VEGF165), a strong pro-angiogenic factor, potentiated the UCB-MSCs multipotency and also resulted in better homing and colonization in liver tissues post-transplantation [ 143 ]. While both native UCB-MSCs and VEGF 165 -encoding UCB-MSC restored liver activity in the ALF rat model, modified stromal cells exhibited more desired therapeutic influences on ALF [ 143 ]. Given that IL-35 plays a pivotal role in Treg-induced immunoregulation, Wang et al. evaluated the therapeutic merits of IL-35 overexpressing MSCs in ALF [ 144 ]. They showed that modified stromal cells migrated to the damaged tissues and considerably reduced the necrosis zones of damaged livers. Moreover, IL-35-MSCs averted hepatocyte apoptosis through down-regulation of the FASL expression by immune cells. They also attenuated IFN-γ levels secreted by immune cells potently via targeting JAK1-STAT1/STAT4 signal pathway [ 144 ]. As described in previous sections, IL-1Ra elicits strong anti-inflammatory and antiapoptotic impacts on immune response in liver failure. Accordingly, Zheng and coworkers showed that transplantation of IL-1Ra-encoding amniotic fluid (AF)-MSCs by the portal vein in the ALF rat model led to reduced mortality as well as ameliorated liver activity [ 145 ].

MSCs-exosome in ALF

Exosomes are small membrane-bound EVs that are produced and then released by numerous types of cells, such as stem/stromal cells, immune cells, or tumor cells. Exosomes are comprised of a myriad of biological components, including proteins, lipids, mRNAs as well as miRNAs as cargo, which can be conveyed to the recipient cells [ 103 ]. Such cargo can adjust physiological cell functions and thereby adapt tissue microenvironment, and also inspire hepatocyte proliferation, reflecting their competencies to be described as a rational therapeutic option in liver diseases, such as ALF (Table 3 ). Reduced levels of miR-20a-5p accompanied with the enhanced level of CXCL8, most eminent neutrophil chemoattractants, are mainly observed in hepatocytes during ALF. But, BM-MSCs-exosome could improve miR-20a-5p expression and conversely attenuate CXCL8 levels in hepatocytes [ 146 ]. Also, systemic injection of UC-MSC-exosome (16 mg/kg) induced liver restoration in the ALF mice model [ 105 ]. It was found that glutathione peroxidase1 (GPX1) enriched exosome-mitigated oxidative stress and apoptosis in the hepatocyte, while the elimination of GPX1 led to the abrogated UC-MSCs-exosome-elicited hepatoprotective impacts in mice [ 105 ]. In addition, UC-MSC-exosomes potently modified the membranous expression of CD154 (or CD40 ligand) in intrahepatic CD4 + T cells, largely contributing to the inflammatory response in the liver [ 147 ]. The suppressive effect on CD154 expression and resultant inflammation was due to the existence of chaperonin containing TCP1 subunit 2 (CCT2) in these exosomes, which targets Ca2 + influx and down-regulates CD154 generation in CD4 + T cells [ 147 ]. In another study, Shao et al. screened the miRNAs in the MSCs-exosomes complicated in inhibition of IL-6-mediated signaling axis in ALF mice model. They showed that miR-455-3p was released by exosomes and efficiently instigated PI3K signaling, and in turn, sustained hepatocyte proliferation [ 106 ]. Also, IL-6 pretreated MSCs or exosomes exhibited higher levels of miR-455-3p compared with native MSCs or their derivative exosome. In fact, miR-455-3p-enriched exosomes suppressed macrophages activation, reduced local liver injury, and also diminish the expression of pro-inflammatory cytokines in vivo [ 106 ]. The miR-455-3p also could constrain activation of HSCs and liver fibrosis upon down-regulation of the heat shock protein (HSP) 47/TGF-β/Smad4 signaling pathway [ 148 ]. Importantly, C-reactive protein (CRP) enriched placenta-derived mesenchymal stromal cells (PD-MSCs)- exosome could up-regulate Wnt signaling pathway as well as angiogenesis in animal hepatocytes by interacting with endothelial cells [ 149 ]. Another study also revealed that rat BM-MSCs-exosome-rich fractionated secretome could bring about a hepatoprotective impact in ALF models mainly caused by diminished oxidative stress [ 150 ]. Similarly, transplantation of exosomes derived from menstrual blood-mesenchymal stromal cells (Men-SCs) that contained a diversity of cytokines, such as intercellular adhesion molecule-1 (ICAM-1 or CD54), angiopoietin-2, Axl, angiogenin, insulin-like growth factor-binding protein 6 (IGFBP-6), osteoprotegerin, IL-6, and IL-8, improved liver function in the ALF animal model [ 151 ]. Treatment resulted in improved survival rates as well as reserved hepatocyte apoptosis. Notably, attenuated numbers of neutrophils and also diminished levels of caspase-3 were evidenced post-transplantation, assuming that Men-SC-exosome can be a substitute treatment to support liver failure [ 151 ]. Pretreatment of UC-MSCs-exosome with TNF-α also enhanced exosome-induced hepatoprotective influence in the ALF mice model [ 107 ]. Pretreated exosomes led to the attenuated serum ALT, AST, and pro-inflammatory cytokines levels and concomitantly down-regulated stimulation of NLRP3 inflammasome. Molecular analysis revealed that miRNA-299-3p up-regulated in TNF-α-primed MSCs-exosome played an eminent role in the amelioration of liver damage in ALF by blocking the NLRP3 pathway [ 107 ]. Apart from its role in liver failure recovery, a miR-299-3p activity as a potent tumor suppressor has been documented in hepatocellular carcinoma by alleviating tumor size and venous infiltration [ 152 ].

MSCs-conditioned medium (CM) could also modify morphological characteristics of hepatocytes in the ALF model. Meanwhile, secretome achieved by cultivating MSCs with low oxygen content (10%) provoked more prominent hepatoprotective influence, and significantly reduced ALT and AST and also pro-inflammatory cytokines serum levels following injection in vivo [ 153 ]. In another study, Li and coworkers evaluated the therapeutic merits of CM from MSCs co-cultured with hepatocytes in the ALF rat model [ 154 ]. The apoptotic cells frequency was lower in CM derived from co-cultured cells than MSCs-CM or hepatocyte-CM. Also, CM derived from co-cultured cells strikingly alleviated liver injury and facilitated liver recovery, indicating the advantages of this strategy for liver failure therapy [ 154 ]. Also, silica magnetic graphene oxide (SMGO) could enhance the hypo protective influences of MSC-CM in ALF in vivo [ 155 ]. Meanwhile, administration of 300 μg/kg SMGO boosted MSC-CM competencies to avert necrosis, apoptosis, and inflammation of hepatocytes. Besides, SMGO therapy up-regulated the expression of VEGF and matrix metalloproteinase-9 (MMP-9) in vitro [ 155 ]. Another report also demonstrated that administration of CM from embryonic stem cell (ESC)-derived MSCs potentiated the proliferation of primary hepatocyte and improved IL-10 secretion from immune cells in vivo [ 156 ]. It appeared that such events might arouse because of the existence of VEGF in ESC-MSC-CM, which affect hepatocytes proliferation and migration, generating new avenues to cure ALF [ 156 ]. Likewise, MSC-CM sustained hepatocytes proliferation, reduced their apoptosis, compromised macrophages infiltration, elevated Th2 and Treg cells population, decreased levels of Th17 cells population, and eventually enabled HSCs death in ALF preclinical model [ 157 ]. The MSC-CM injection caused glycogen synthesis and storage recovery and also ameliorated ALF with no effect on Th1 cells [ 157 ]. Also, CM achieved from either amniotic fluid (AF)-MSCs or hepatic progenitor-like (HPL) cells derived from AF-MSCs thanks to the presence of IL-10, IL-1Ra, IL-13, and IL-27 stimulated liver recovery in the mice model with ALF [ 158 ].

MSCs in liver-associated conditions (clinical trials)

Several clinical trials have been accomplished or are ongoing to address the safety, feasibility and efficacy of MSCs therapy in liver-associated conditions, most frequently in liver failure and liver cirrhosis (Table 4 , Fig.  2 ). BM-MSCs and UC-MSCs are most commonly used types of cells. Of course, there is still no definite standard for which source of MSCs should be applied for clinical use. It seems that UC-MSCs are preferred for liver failure treatment as a result of higher differentiation capability. Also, the immunogenicity of UC-MSCs is lower than that of BM-MSCs [ 159 , 160 ]. Hence, autologous BM-MSCs and allogeneic UC-MSCs are highly preferred. On the other hand, poor proliferation, anti-inflammatory and self-renewal ability impedes AT-MSCs application in clinic [ 161 ]. Although intravenous injection is most used route, intraportal administration is evidently the optimal route for MSC therapy in liver-associated conditions due to the faster engraftment and the prohibited off-target accumulation. However, we must assess patients’ conditions and the potential risk of applying a particular route before choosing the administration route.

Clinical trials based on mesenchymal stromal cells (MSCs) therapy in liver-associated conditions registered in ClinicalTrials.gov (November 2021). The schematic demonstrates clinical depending on the study phase ( A ), study status ( B ), MSCs source ( C ), study location ( D ), participant number ( E ), and condition ( F )

Liver failure

A study of the safety and preliminary efficacy of UC-MSC transplantation (3 times at 4-week intervals) was carried out by Ming and colleagues [ 162 ]. They showed that the intervention had no unwanted effects, while attenuated total bilirubin and ALT levels, prolonged survival rate, and finally ameliorated liver functions, as evidenced by improved serum albumin, and prothrombin activity [ 162 ]. As well, intrasplenic and intrahepatic administration of autologous BM-MSCs derived hepatocyte inspired short-term amelioration in patient’s ascites, lower limb edema, and serum albumin [ 163 ]. Of course, defining the life span of the transplanted cells, and also determining the presence of long-term side effects is urgently required [ 163 ]. Moreover, another trial, which was accomplished from 2010 to 2013, indicated that systemic administration of allogeneic BM-MSCs could exert therapeutic benefits in patients suffering from HBV-related LF [ 164 ]. Meanwhile, stromal cell therapy augmented serum total bilirubin and ultimately promoted the 24-week survival rate by stimulating liver rescue concomitant with lessening the occurrences of stern infections compared with the control group (16.1% versus 33.3%) [ 164 ]. Likewise, other trials also exhibited that autologous BM-MSC transplantation was safe for chronic HBV-induced LF patients, as shown by the incidence of no serious intervention-related events and carcinoma during 192 weeks follow-up [ 165 ]. Also, the short-term outcome was promising; however, long-term efficacy was not clearly amended [ 165 ].

Liver cirrhosis

Cirrhosis is a late-stage liver disease in which healthy liver tissue is substituted with scar tissue and the liver is perpetually damaged. Liver transplantation is a standard therapeutic plan aiming to treat liver cirrhosis patients [ 166 ]. Meanwhile, MSCs have recently been noted as a possible therapeutic option to partially ameliorate liver function in this condition as a result of their appreciated antifibrotic and immunoregulatory attributes [ 55 ].

A phase 1 trial on 4 patients with decompensated liver cirrhosis verified the safety and feasibility of MSCs therapy [ 167 ]. Moreover, the life quality of all patients was ameliorated post-transplantation concerning the mean physical and mental component scales [ 167 ]. In primary biliary cirrhosis patients, UC-MSC injection by peripheral vein (3 times at 4-week intervals) exhibited no serious untoward effects [ 168 ]. Also, intervention caused a robust reduction in serum alkaline phosphatase (ALP) and γ-glutamyltransferase (GGT) levels compared to the control group during 4 years follow-up. Notwithstanding, no alteration was observed in levels of ALT, AST, total bilirubin, albumin, INR, or the prothrombin time activity. Thereby, comprehensive randomized controlled cohort trials are justified to authorize the clinical merits of UC-MSC transplantation [ 168 ]. In addition, injection of autologous BM-MSCs led to a partial amelioration of liver function in 25 Egyptian patients suffering from HCV-triggered liver cirrhosis, as evinced by improved prothrombin activity and serum albumin levels along with reduced bilirubin level [ 169 ]. In a similar condition, Amin et al. found that intrasplenic administration of autologous BM-MSCs potentiated liver function with attenuation in total bilirubin, AST, ALT, prothrombin time (PT), and also INR levels [ 170 ]. Autologous BM-MSCs therapy also inspired an improvement in liver function among HBV-related liver cirrhosis patients following transplantation [ 171 ]. This trial was conducted in 56 patients with HBV-induced liver cirrhosis, and results showed an enhancement in Treg/Th17 ratio post-transplantation during 24-week follow-up [ 171 ]. Consistently, mRNA levels of forkhead box protein P3 (FOXP3), an eminent Treg-associated transcription factor, strikingly were diminished, whereas retinoic acid-related orphan receptor gamma t (RORγt) expression levels which are tightly in association with Th17 cells were reduced. Further, the intervention resulted in an enhancement in TGF-β levels, while IL-17, TNF-α, and IL-6 were significantly decreased following transplantation [ 171 ]. In contrast to several cited trials implying that the autologous MSC therapy can be a safe and effective alternative for patients with liver cirrhosis [ 172 , 173 ], Mohamadnejad et al. noted that MSC infusion by peripheral vein had no advantageous result in cirrhotic patients [ 174 ]. Overall, large-scale studies are required to achieve reliable results concerning MSCs therapy in liver cirrhosis.

Potential risks of MSC transplantation

The treatment of liver dysfunctions through MSCs has been the central aim of several clinical and preclinical investigations. In this context, a few issues need to be dealt with cautiously (e.g., the possible emergence of carcinogenesis and the transmission of the virus). Different growth factors can be secreted by MSCs, and this may stimulate the growth of tumor cells and angiogenesis [ 175 , 176 ]. The past experimental investigations showed that the number of passages is a defining factor in rendering a tissue malignant or cancerous. Studies show that chromosome abnormalities may occur after more than three passages in the MSCs of mice [ 177 , 178 ]. Moreover, MSCs are likely to experience telomeric deletions after a multitude of passages. Despite the lack of any clinical reports on the malignant transformation of human MSCs, the follow-up period was not long enough for the formation of a tumor for most of them [ 179 ]. As a result, there need to be more studies on chromosomal integrity before MSCs transplantation to make sure that the procedure is completely safe.

Contrary to autotransplantation, allotransplantation can pose the danger of the spread of the virus to the patients [ 180 ]. Even though the spread of parvovirus B19 into BM cells was observed in vitro, there is no confirmed case of parvovirus B19-positive MSC-related viremia in humans. Yet, we do not know the spread of the herpes simplex virus (HSV) and cytomegalovirus (CMV) via MSCs in vivo. Owing to these facts, recipients, and donors of MSC are recommended to be screened for parvovirus B19, HSV, and CMV, as immunosuppressed patients are likely to catch infectious [ 181 ].

Enhancing the quantity of MSCs-secreted molecules

Now, restricted secretion of soluble mediators, such as exosome, from parental MSCs fences their wide-ranging application in clinics. Following some passages, MSCs mainly demonstrates abrogated competence to produce and then release soluble factor. Recent studies have indicated that tangential flow filtration (TFF) system-based tactics support the secretion of greater levels of vesicles from origin stromal cells than vesicle isolation by ultracentrifuge [ 182 ]. Further, ultrasonication of MSC-derived extracellular vesicles could improve their yields up to 20-fold [ 183 ]. Other proofs are indicating that three dimensional (3D) culture may facilities the incessant production of MSC-derived exosome [ 184 , 185 ]. Cultivation of MSCs in 3D cultures together with conventional either differential ultracentrifugation or TFF also could engender a higher quantity of MSCs-derived secretome [ 186 ]. Also, MSCs culture on particular biomaterials, such as alginate hydrogel [ 187 ] and avitene ultrafoam collagen ease generation of exosome with higher quantity and also potency [ 188 ]. As well, pretreatment of MSCs with hypoxia or various molecules, in particular cytokines or chemokines (e.g., IFN-γ, TNFα, IL-1β, IL-6, and TGF-β), gives largely rise to the secretion of vesicles with greater regenerative competencies [ 189 , 190 , 191 ].

Conclusion and future direction

Some investigations in preclinical models of liver diseases, such as ALF, have verified the MSC's unique competence to establish hepatocyte in vivo. Nonetheless, it seems that the therapeutic merits of MSCs largely rely on their aptitudes to secrete a myriad of factors, more importantly, cytokines, growth factors, and miRNAs, facilitating liver recovery. During last two decades, various clinical trials have been conducted to evaluate the capability of MSCs therapy in liver-associated conditions, such as ALF (Table 4 , Fig.  2 ); however, achieved outcomes are quite inconsistent. Given that autologous MSCs derived from elder patients or patients with obesity experienced abrogated proliferation and differentiation capability, using allogeneic cells in some cased is urgently required. In this circumstance, screening recipients and donors of MSC for parvovirus B19, HSV, and CMV are of paramount importance. Taken together, the providing of a universal MSC quality standard evaluation system is required.

To determine the mechanism contributed to MSCs therapy, it is urgently required to determine the protein, DNA and RNA secreted by MSCs. The proteomics and transcriptomics can play a pivotal role in evaluating the underlying mechanism. Notably, improving the frequency of cells homing to the damaged liver is the central point to potentiate the therapeutic impacts of MSCs. In fact, investigation of the homing attributes of MSCs is of paramount importance to augment the effective therapeutic quantity of such cells. In published clinical results, MSCs have been administrated into patients by several available routes, more frequently intravenous routes followed by intrahepatic injection (e.g., by the portal vein and hepatic artery). Also, intrasplenic injection has been applied in a few studies. Based on findings, a remarkable number of cells are trapped in the lungs upon systemic injection and thereby did not move to the liver afterward. Hence, finding better administration route is recommended to achieve significant outcome in vivo. Meanwhile, a study indicated that intraportal injection was more effective than hepatic intra-arterial injection and also intravenous injection to restore liver injury in vivo [ 123 ]. As well, it has been shown that portal vein injection has superiority over intrasplenic injection [ 192 ]. On the other hand, other reports exhibited that injection by the hepatic artery was not beneficial for the transdifferentiation of MSCs.

Among the recent clinical trials concerning the MSCs therapy for liver diseases (e.g., liver failure) treatment, the total number of MSCs employed was from 10 6 to 10 9 , irrespective of which method was applied to deliver the dose. The large range of doses applied is difficult to explicate as there are few reports including comparisons of several doses in the same clinical trial. Nonetheless, it seems that as few as 1 × 10 7 cells can be helpful based on recent published results.

In sum, although clinical trials have evidenced the safety and modest efficacy of short-term application of MSCs, further trials are warranted before MSCs application in clinical to treat ALF and other liver-associated conditions for optimizing administration routes as well as dosses.

Availability of data and materials

Not applicable.

Abbreviations

Mesenchymal stromal cells

Acute live failure

Hepatocyte growth factor

Tumor necrosis factor α

Interferon gamma

Vascular endothelial growth factor

Bone marrow

Umbilical cord

Adipose tissue

Transforming growth factor β

Aspartate aminotransferase

Alanine aminotransferase

Interleukin

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Samin Shokravi & Sima Marzban

I.M. Sechenov First Moscow State Medical University (Sechenov University), Moscow, Russian Federation

Vitaliy Borisov

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Shokravi, S., Borisov, V., Zaman, B.A. et al. Mesenchymal stromal cells (MSCs) and their exosome in acute liver failure (ALF): a comprehensive review. Stem Cell Res Ther 13 , 192 (2022). https://doi.org/10.1186/s13287-022-02825-z

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DOI : https://doi.org/10.1186/s13287-022-02825-z

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