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Article Contents

In this issue, acknowledgements, disclosures, plant stem cells: the source of plant vitality and persistent growth.

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Makoto Hayashi, Ari Pekka Mähönen, Hitoshi Sakakibara, Keiko U Torii, Masaaki Umeda, Plant Stem Cells: The Source of Plant Vitality and Persistent Growth, Plant and Cell Physiology , Volume 64, Issue 3, March 2023, Pages 271–273, https://doi.org/10.1093/pcp/pcad009

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Plants have amazing vitality and persistence. Some tree species can live for thousands of years, and even when cut down, new sprouts emerge from the stump continuing their life. Similarly, if a horsetail is cut and fragmented, it is also capable of regenerating and flourishing from a remaining rhizome. Such features are based on the remarkable characteristics of plant stem cells. Plants are able to maintain pluripotency in stem cells generated during embryogenesis, and even after their differentiation, the cells can reprogram themselves and regenerate the whole plant body by acquiring pluripotency in response to stresses, such as wounding. Although humankind depends on the productive capacity of plants to meet various needs such as food, raw materials and maintenance of the global environment, we are yet to gain an understanding of the regulatory systems that generate their robust vitality. In other words, the elucidation of the molecular basis of plant vitality is one of the central issues not only in plant and agricultural sciences but also in life sciences.

The history of stem cell study in plants is relatively young compared to that in animals. In plants, the dividing cell population containing stem cells is called a meristem and the maintenance and regulatory mechanisms of its activity have been studied. Nonetheless, our understanding of the intrinsic properties of plant stem cells had been somewhat limited due to difficulties in accessing these deeply embedded tissues. However, thanks to recent advances in molecular genetics and single cell technologies, it is now possible to study stem cell characteristics more deeply.

This special issue explores the latest research into plant stem cells. The idea for this special issue was borne from a consortium research project ‘Principles of pluripotent stem cells underlying plant vitality’, which was conducted from 2017 to 2021 and supported by a Grant-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Sports, Science and Technology, Japan. The project aimed to understand the characteristics of plant stem cells through a multifaceted approach to study the temporal and spatial control of stem cell proliferation and generation and the mechanisms underpinning the maintenance of pluripotency and genome homeostasis. The ultimate goal was to understand persistence and vitality characteristics of plants to enable sustainable organogenesis and regeneration, as also reflected in the pages of this special issue.

This special issue includes four mini-reviews and four original articles focusing on different aspects of plant stem cells, briefly summarized later.

Shoot stem cells are the source of all post-embryonic aerial organs. An elaborate regulatory system is required to ensure that plant stem cells maintain their correct status during growth and development. Wang et al. (2023) summarize recent breakthroughs in studies of genetic circuits controlling the fate of shoot stem cells, namely, arrest, senescence and death. More specifically, they illustrate a working model for shoot apical meristem arrest (or end-of-flowering) under the FRUITFULL–APETALA2 pathway ( Balanzà et al. 2018 ) and propose a model for stem cell death controlled by dynamic changes in reactive oxygen species.

Plants continuously form branches to increase their photosynthetic capacity and expand their territories. Shoot branches are derived from axillary meristems initiated at the leaf axils, and the continuous formation of new axillary meristems allows for the plastic expansion of highly branched shoot systems. Axillary meristems arise from the division of boundary domain cells at the leaf base, but how axillary meristems are established de novo remains to be fully elucidated ( Nicolas and Laufs 2022 ). Yang et al. (2023) summarize recent progress in understanding the regulation of axillary meristem initiation, focusing on the key transcription factors, phytohormones and microRNAs involved. The illustration of a working model helps us to understand sequential processes leading to axillary meristem initiation, which constitutes an excellent system for determining stem cell fate and de novo meristem formation.

In both shoots and roots, persistent growth and organogenesis depend on the continued activity of meristems located in their apices. To establish persistency, a key system is the separation of cells into specific domains with different activities, called zonation. In roots, a dynamic equilibrium is reached in which cell division in the stem cell niche and meristem and cell differentiation in the elongation/differentiation region are balanced, which stabilizes the number of dividing cells and maintains the position of the transition zone ( Salvi et al. 2020 , Svolacchia et al. 2020 ). Shtin et al. (2023) show that the mutual inhibitory regulation between the PLETHORA (PLT) and the ARABIDOPSIS RESPONSE REGULATOR (ARR) transcription factors is sufficient for root zonation, separating cell division and cell differentiation during organogenesis. Specifically, they demonstrated that ARR1 suppresses PLT activities and that PLTs suppress ARR1 and ARR12 by targeting their proteins for degradation via the KISS ME DEADLY 2 F-box protein. These findings provide new insight into the complex process of root zonation.

Plant cells, including highly differentiated cells, have a remarkable capacity for reprogramming, resulting in the de novo generation of a whole plant. In the past two decades, extensive studies using the model plant Arabidopsis have uncovered the basic molecular scheme for plant regeneration ( Mathew and Prasad 2021 ). However, many important questions, such as how plant cells retain both differentiated status and developmental plasticity, still remain. Morinaka et al. (2023) provide an overview of the representative modes of plant regeneration and key factors revealed in studies of Arabidopsis and re-examine historical tissue culture systems that enable us to investigate the molecular details of cell reprogramming in highly differentiated cells.

The precise control of cell growth and proliferation is essential for the appropriate development of multicellular organisms, including plants. Critical regulatory factors controlling cell division and growth have been identified, but the mechanisms underlying cell type–specific cell growth and proliferation are still poorly understood. Ta et al. (2023) characterized a rice mutant with reduced mitotic activity, which is defective in the progression of embryogenesis. The causal gene encodes a member of the MO25A family of proteins that have pivotal functions in cell proliferation and polarity in animals, yeasts and filamentous fungi. Functional analysis of MO25A in the moss Physcomitrium patens showed that P. patens MO25A takes part in cell tip growth and the initiation of cell division in stem cells, suggesting that MO25A proteins have a conserved function that controls cell proliferation and growth across all kingdoms.

Some plant cell types are generated de novo through stem-cell-like precursors. During stomatal development of Arabidopsis , the sequential process of cell division and differentiation is governed by the key transcription factors, such as MUTE and FAMA, which switch the cell cycle mode from asymmetric division to symmetric division and terminate the cell cycle. This sequential process occurs within a single round of the cell cycle; however, it remains elusive whether the cell cycle restricts the expression of these transcription factors. Zuch et al. (2023) investigated the expression patterns of MUTE and FAMA during the cell cycle and found that MUTE expression is gated by the cell cycle. Moreover, they revealed that, in the absence of MUTE, the G1 phase is prolonged as the meristemoids reiterate asymmetric cell divisions. This study highlights a mechanism for the eventual G1 arrest of an uncommitted stem-cell-like precursor.

The vascular system transports water and nutrient ions and assimilates throughout the plant body. Key factors and the regulatory networks of primary and secondary vascular development have been identified ( Haas et al. 2022 ). However, the complexity of the vascular system, which is composed of a variety of cells including xylem and phloem cells, makes it difficult to analyze vascular development and distinguish between vascular stem cells and developing xylem and phloem cells. Shimadzu et al. (2023) summarize recent findings on the establishment and maintenance of vascular stem cells, focusing on recent technical advances that enable cell type–specific analysis during vascular development.

Grafting is a horticultural technique that physically connects two individual plants of different genetic backgrounds to create or enhance properties such as abiotic stress resistance. During this process, callus formation at the graft junction facilitates organ attachment and vascular reconnection. Ikeuchi’s group recently identified WUSCHEL-RELATED HOMEOBOX13 (WOX13) as an essential regulator of organ grafting ( Ikeuchi et al. 2022 ), but how callus formation is differentially regulated at each cut end remained unsolved. In this issue, Tanaka et al. (2023) report that differential auxin signaling between the top and bottom cut ends of grafted stems is responsible for the commonly observed asymmetric callus formation. Specifically, they found that this process is regulated by differential auxin accumulation and that expression of auxin-responsive genes, including WOX13 , preferentially occurs in the top part of the graft. Their findings provide insight into the role of auxin signaling in organ attachment during the grafting process.

Finally, we hope that the papers in this special issue help readers to update their current understanding of plant stem cells and provide new ideas for future conceptual breakthroughs in plant stem cell biology.

Ministry of Education, Culture, Sports, Science and Technology, Japan [Grants-in-Aid for Scientific Research on Innovative Areas (Principles of Pluripotent Stem Cells Underlying Plant Vitality, 17H06470 and 22H04904) to M.U.].

We thank Professor Wataru Sakamoto, Editor-in-Chief, Plant and Cell Physiology , for providing the opportunity for this special issue. We would like to acknowledge the authors and reviewers who have greatly contributed to this issue.

The authors have no conflicts of interest to declare.

Balanzà   V. , Martínez-Fernández   I. , Sato   S. , Yanofsky   M.F. , Kaufmann   K. , Angenent   G.C. , et al.  ( 2018 ) Genetic control of meristem arrest and life span in Arabidopsis by a FRUITFULL-APETALA2 pathway . Nat. Commun.   9 : 565.

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Ikeuchi   M. , Iwase   A. , Ito   T. , Tanaka   H. , Favero   D.S. , Kawamura   A. , et al.  ( 2022 ) Wound-inducible WUSCHEL-RELATED HOMEOBOX 13 is required for callus growth and organ reconnection . Plant Physiol.   188 : 425 – 441 .

Mathew   M.M. and Prasad   K. ( 2021 ) Model systems for regeneration: Arabidopsis . Development   148 : dev195347.

Morinaka   H. , Coleman   D. , Sugimoto   K. and Iwase   A. ( 2023 ) Molecular mechanisms of plant regeneration from differentiated cells: approaches from historical tissue culture systems . Plant Cell Physiol.   64 : 308 – 315 .

Nicolas   A. and Laufs   P. ( 2022 ) Meristem initiation and de novo stem cell formation . Front. Plant Sci.   13 : 891228.

Salvi   E. , Rutten   J.P. , Di Mambro   R. , Polverari   L. , Licursi   V. , Negri   R. , et al.  ( 2020 ) A self-organized PLT/Auxin/ARR-B network controls the dynamics of root zonation development in Arabidopsis thaliana . Dev. Cell.   53 : 431 – 443 .

Shimadzu   S. , Furuya   T. and Kondo   Y. ( 2023 ) Molecular mechanisms underlying the establishment and maintenance of vascular stem cells in Arabidopsis thaliana . Plant Cell Physiol.   64 : 285 – 294 .

Shtin   M. , Polverari   L. , Svolacchia   N. , Bertolotti   G. , Unterholzner   S.J. , Di Mambro   R. , et al.  ( 2023 ) The mutual inhibition between PLETHORAs and ARABIDOPSIS RESPONSE REGULATORs controls root zonation . Plant Cell Physiol.   64 : 328 – 335 .

Svolacchia   N. , Salvi   E. and Sabatini   S. ( 2020 ) Arabidopsis primary root growth: let it grow, can’t hold it back anymore!   Curr. Opin. Plant Biol.   57 : 133 – 141 .

Ta   K.N. , Yoshida   M.W. , Tezuka   T. , Shimizu-Sato   S. , Nosaka-Takahashi   M. , Toyoda   A. , et al.  ( 2023 ) Control of plant cell growth and proliferation by MO25A, a conserved major component of the Mammalian Sterile20-like kinase pathway . Plant Cell Physiol.   64 : 347 – 362 .

Tanaka   H. , Hashimoto   N. , Kawai   S. , Yumoto   E. , Shibata   K. , Tameshige   T. , et al.  ( 2023 ) Auxin-induced WUSCHEL-RELATED HOMEOBOX13 mediates asymmetric activity of callus formation upon cutting . Plant Cell Physiol.   64 : 316 – 327 .

Wang   Y. , Shirakawa   M. and Ito   T. ( 2023 ) Arrest, senescence and death of shoot apical stem cells in Arabidopsis thaliana . Plant Cell Physiol.   64 : 295 – 301 .

Yang   T. , Jiao   Y. and Wang   Y. ( 2023 ) Stem cell basis of shoot branching . Plant Cell Physiol.   64 : 302 – 307 .

Zuch   D.T. , Herrmann   A. , Kim   E.-D. and Torii   K.U. ( 2023 ) Cell cycle dynamics during stomatal development: window of MUTE action and ramification of its loss-of-function on an uncommitted precursor . Plant Cell Physiol.   64 : 336 – 346 .

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Plant stem cells and their applications: special emphasis on their marketed products

Affiliations.

• 1 Amity Institute of Pharmacy, Amity University, Noida, Uttar Pradesh 201313 India.
• 2 Department of Botany, Ege University, Izmir, Turkey.
• PMID: 32550110
• PMCID: PMC7275108
• DOI: 10.1007/s13205-020-02247-9

Stem cells are becoming increasingly popular in public lexicon owing to their prospective applications in the biomedical and therapeutic domains. Extensive research has found various independent stem cell systems fulfilling specific needs of plant development. Plant stem cells are innately undifferentiated cells present in the plant's meristematic tissues. Such cells have various commercial uses, wherein cosmetic manufacture involving stem cell derivatives is the most promising field at present. Scientific evidence suggests anti-oxidant and anti-inflammatory properties possessed by various plants such as grapes ( Vitis vinifera ), lilacs ( Syringa vulgaris ), Swiss apples ( Uttwiler spatlauber ) etc. are of great importance in terms of cosmetic applications of plant stem cells. There are widespread uses of plant stem cells and their extracts. The products so formulated have a varied range of applications which included skin whitening, de-tanning, moisturizing, cleansing etc. Despite all the promising developments, the domain of plant stem cells remains hugely unexplored. This article presents an overview of the current scenario of plant stem cells and their applications in humans.

Keywords: Anti-ageing; Cosmetics; Plant stem cells; Skincare; Stem cell extract.

© King Abdulaziz City for Science and Technology 2020.

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• Open access
• Published: 14 September 2022

The potential of plant extracts in cell therapy

• Caifeng Li 1 ,
• Zhao Cui 2 ,
• Shiwen Deng 1 ,
• Peng Chen 1 , 3 ,
• Xianyu Li 1 &
• Hongjun Yang 1 , 3  

Stem Cell Research & Therapy volume  13 , Article number:  472 ( 2022 ) Cite this article

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Cell therapy is the frontier technology of biotechnology innovation and the most promising method for the treatment of refractory diseases such as tumours. However, cell therapy has disadvantages, such as toxicity and poor therapeutic effects. Plant extracts are natural, widely available, and contain active small molecule ingredients that are widely used in the treatment of various diseases. By studying the effect of plant extracts on cell therapy, active plant extracts that have positive significance in cell therapy can be discovered, and certain contributions to solving the current problems of attenuation and adjuvant therapy in cell therapy can be made. Therefore, this article reviews the currently reported effects of plant extracts in stem cell therapy and immune cell therapy, especially the effects of plant extracts on the proliferation and differentiation of mesenchymal stem cells and nerve stem cells and the potential role of plant extracts in chimeric antigen receptor T-cell immunotherapy (CAR-T) and T-cell receptor modified T-cell immunotherapy (TCR-T), in the hope of encouraging further research and clinical application of plant extracts in cell therapy.

Introduction

As the frontline of biotechnological innovation, cell therapy has a significant influence on medical treatment and provides new sights for difficult diseases [ 1 ]. Cell therapy mainly includes stem cell therapy and immune cell therapy. Stem cells can be acquired from embryonic tissue, foetal tissue, adult organism, and induced pluripotent stem cells (iPSCs) [ 2 , 3 ]. According to developmental stage, stem cells include pluripotent stem cells (PSCs) and adult stem cells. Among them, PSCs include embryonic stem cells (ESCs) and iPSCs [ 3 ], which are pluripotent. However, ESCs have ethical issues, and the probability of reproductive cloning and tumour formation limited the application of iPSCs [ 3 ]. Adult stem cells overcome these problems of PSCs, which contain mesenchymal stem cells (MSCs), neural stem cells (NSCs), and adipose-derived stem cells (ASCs), among others, and have significant therapeutic effects in cardiovascular, neurological, skeletal, and autoimmune diseases [ 4 , 5 ]. In addition, immune cell therapy, represented by chimeric antigen receptor T-cell immunotherapy (CAR-T), and T-cell receptor modified T-cell immunotherapy (TCR-T), has shown potential efficacy in multiple myeloma and other haematologic malignancies as well as in some solid tumours [ 6 ]. Cell therapies have a large latent capacity under the development of modern science and technology. However, cell therapy itself still has deficiencies in practice, such as side effects, inflammatory factor storms caused by excessive immune responses and poor effects in some patients. As a result, combining it with other drugs may be a way to solve this problem. Additionally, protein cytokines and antibodies have been widely used in cell culture and clinical treatment, with disadvantages such as toxicity, and high cost. Therefore, at present, looking for alternative plant extracts that can be used as growth factors and adjuvant therapies in cell therapy is a promising approach [ 7 ].

Hormesis effects on stem cells have been observed, mainly in the use of drugs (e.g. metformin, atorvastatin, and isoproterenol), dietary supplements/medicinal plant extracts (e.g. berberine), and endogenous drugs (e.g. estrogen) [ 8 , 9 ]. Among them, plant extracts are an important source of bioactive small molecules, which are mainly derived from herbal plants, and include flavonoids, alkaloids, polysaccharides, volatile oils, etc. Plant extracts play an important role in disease treatment, especially in cancer and infectious diseases. Herbal therapy is a traditional medical practice that has long been used to treat a variety of diseases. Herbal medicine is safe and affordable [ 7 ]. It is a promising alternative approach with a significant effect on alleviating patient disease. Therefore, studying the effects of plant extracts in cell therapy and digging out active plant extracts in herbal medicines can provide guidance for adjuvant therapy combined with cell therapy.

The application of plant extracts in cell therapy has gradually increased with the rapid development of cell therapy. For example, the increased therapeutic effects of stem cells induced by plant extracts have been reported in studies of Alzheimer’s disease [ 10 ], chronic kidney disease [ 11 ], and stroke [ 12 ], and the therapeutic effects of immune cells induced by plant extracts have been reported in diseases such as non-small-cell lung cancer and liver cancer [ 13 , 14 ]. This review describes the effects of plant extracts, mainly herbal extracts, in cell therapy approaches and clarifies the potential mechanisms of action when possible.

The effect of plant extracts on stem cell therapy

The effect of plant extracts on mscs.

MSCs exist in almost all postnatal human tissues. The major sources of adult MSCs are mainly from bone marrow, adipose tissue, etc. [ 7 , 15 ]. Compared with ESCs, MSCs are easier to isolate and culture in vitro, and more importantly, there are fewer ethical issues. Furthermore, due to their HLA-DR-negative feature, MSCs do not have immunogenic in therapy [ 7 ]. MSCs are characterized by different sources, isolation methods, and epigenetic changes during growth. They can be differentiated into osteocytes, neurons, and angiogenesis, through stimulation with plant extracts (Fig.  1 ).

Sources of MSCs and their proliferation, differentiation, angiogenesis, antilipogenesis, and antioxidant stress effects stimulated by plant extracts

Proliferation effect

Many plant extracts, such as Foeniculum vulgare [ 16 ] , Ferula gummosa [ 17 ], amentoflavone ( Selaginella tamariscina (P. Beauv.) Spring) [ 18 ], gastrodin ( Gastrodia elata Bl.) [ 19 ], and resveratrol ( Polygonum cuspidatum Sieb. et Zucc.) [ 20 ], can significantly increase bone marrow-derived human MSCs (BM-hMSCs) proliferation. In addition, ginsenoside Rg1, which is an effective compound in Panax ginseng , Panax notoginseng , and American ginseng, can also promote cell proliferation [ 21 ]. Apple ethanol extract promotes proliferation of hASCs and human cord blood-derived MSCs via ERK signalling [ 22 ]. Tinospora cordifolia and Withania somnifera are traditional Ayurveda medicinal materials in India that are reported to improve cell proliferation ability and activity, as well as reduce cell apoptosis and postpone aging [ 23 ]. ZD-I is a prescription composed of seven traditional Chinese medicines (TCM). It has stimulatory effects on the proliferation of hMSCs [ 24 ]. Viscum album induces primitive placenta-derived MSCs (PDSCs) with remarkable proliferative properties through autophagy mechanism. Specifically, Viscum album can regulate the cell cycle to make PDSCs self-renewal and regulate the induction of survival factors, apoptosis and autophagy to reduce cell death [ 25 ].

Differentiation effect

Osteogenic effects, osteogenic effects via transcription factors.

Foeniculum vulgare, Ferula gummosa, and amentoflavone can significantly increase the alkaline phosphatase (ALP) activity of hMSCs and promote BM-hMSC differentiation into osteoblasts [ 16 , 17 , 18 ]. Dipsacus asper and its ingredients hedraganin-3- O -(2- O -acetyl)-α- l -arabinopyranoside enhance osteoblastic differentiation not only by inducing ALP activity but also by inducing bone sialoprotein and osteocalcin expression [ 26 ]. Moreover, Fructus Ligustri Lucidi effectively activated ALP, reduced the osteogenic differentiation time of MSCs, and up-regulated the expression of osteogenic related factors such as catenin, BMP2, cyclin D1, membrane matrix metalloproteinase, osteoprotegerin and T-box 3 [ 27 ]. Poncirin ( Poncirus trifoliata (L.) Raf) [ 28 ], Panax notoginseng saponins [ 29 , 30 ], and naringin ( Citrus grandis ) [ 31 , 32 ] decreased peroxisome proliferator-activated receptor γ (PPARγ) 2 mRNA levels, while Panax notoginseng saponins raised the levels of ALP, Cbfa 1, OC, BSP, OPG, β-catenin, and cyclin D1. Harmine ( Peganum harmala L.) increased ALP activity and up-regulated osteocalcin expression. Moreover, harmine can up-regulate osterix [ 33 ]. The combination of epigallocatechin-3-gallate [ 34 ] and bone inducer can up-regulate BMP2 and enhance bone formation. Gastrodin [ 19 ] improved ALP, OCN, COL I, and OPN while reducing ROS. Fucoidan enhanced osteogenic specific marker genes, such as ALP, osteopontin, type I collagen, Runx2, and osteocalcin in ASCs [ 35 ]. Quercetin ( Sophora flavescens Ait.) can increase Osx, Runx2, BMP2, Col1, OPN and OCN, and enhance osteogenic differentiation [ 36 ]. BuShenNingXin decoction (BSNXD) up-regulated ALP and collagen type I, osteocalcin, Runx2, and osterix. Furthermore, BSNXD was shown to reduce the quantity of adipocyte and PPARγ mRNA [ 37 ]. A summary table of plant extracts that stimulate osteogenesis of MSCs is shown in Additional file 1 : Table S1.

Osteogenic effects through Wnt signalling pathways

Flavonoids of epimedii( Epimedium brevicornum Maxim., etc.) were found to increase the rates of osteogenic activity through the BMP or Wnt-signalling pathway [ 38 ]. In addition, Angelica sinensis polysaccharide can enhance the osteogenic differentiation of rat BM-MSCs cultured in high-sugar and guide bone regeneration in type 2 diabetes animal model which chained to the Wnt/β-catenin signalling pathway [ 39 ]. Ginkgo biloba and its main component ginkgolide B accelerate osteoblast differentiation and the formation of bone via Wnt/β-Catenin signalling [ 40 ] (Fig.  2 ). Berberine ( Coptis chinensis Franch.) [ 41 ] and salvianolic acid B ( Salvia miltiorrhiza Bge.) [ 42 , 43 ] promote osteogenesis in BM-MSCs through Wnt/β-catenin signalling and strengthen Runx2 expression. Salvianolic acid B influences the ERK signalling pathway and lower PPARγ mRNA, accelerating the osteogenesis of MSCs. The osteogenic-related genes can be strengthened under the induction of naringin, and the expression of Notch1 can be up-regulated at the same time, and activation Wnt signalling activation [ 44 ].

Plant extracts that affect MSCs osteogenesis by regulating intracellular signalling pathways. Ginkgolide B, Panax notoginseng saponins, berberine, and salvianolic acid B regulate axin, β-catenin, and TCF in the Wnt signalling pathway; Ginkgo biloba , harmine, silibinin, genistein, and Ligusticum chuanxiong regulate BMP, Runx2 and Smad 1/5/8 in the BMP signalling pathway; Resveratrol, icariin, amentoflavone, quercetin, and fucoidan regulate p38, ERK1/2, and JNK in the MAPK signalling pathway

Osteogenic effects through BMP signalling pathways

Ginkgo biloba has been found to enhanced Runx2 expression and regulated BMP4 in BMP signalling [ 45 ]. Moreover, harmine [ 33 ], silibinin( Silybum marianum (L.) Gaertn.) [ 46 ], and genistein( Genista tinctoria Linn., etc.) [ 47 ] activate the BMP and Runx2 pathways. Duhuo Jisheng decoction and its effective component Ligusticum Chuanxiong can activate Smad 1/5/8 and ERK signalling, increase the osteogenic effect of MSCs, and improve BMP-2 and Runx2 [ 48 ] (Fig.  2 ).

Osteogenic effects through MAPK signalling pathways

Most of the plant extracts used in the study of MSCs osteogenesis are TCM monomers. Icariin ( Epimedium brevicornum Maxim., etc.), amentoflavone and quercetin promoted osteogenesis via the JNK and p38 MAPK pathways (Fig.  2 ). Icariin also phosphorylates ERK, and stimulates PI3K-AKT-eNOS-NO-cGMP-PKG pathway in bone marrow stromal cells [ 18 , 49 , 50 ]. Quercetin is a flavonoid that can also activate ERK signalling pathways, decrease the aging and oxidative stress in MSCs, and promote osteogenic differentiation [ 51 , 52 ]. Fucoidan can induce osteogenic differentiation, activate ERK and JNK mainly through BMP2 Smad 1/5/8 signalling, and regulate osteogenic differentiation markers [ 53 , 54 ]. One study showed that resveratrol enhanced cell renewing by inhibiting cell aging at a low concentration, while it inhibits cell self-renewal by up-regulating cell senescence, doubling time, and S-phase arrest at a high concentration. In addition, it can stimulate MSCs and promote osteoblast differentiation by acting on ER-dependent mechanisms and activating ERK1/2 [ 20 , 55 , 56 ].

Neurogenic effects

Mucuna gigantea grows natively in Hawai ‘i. It was recorded that it can be used to treat kampavata (excitatory paralysis) [ 57 ]. Mucuna gigantea can promote proliferation feature, nestin, and β-III tubulin mRNA expression in MSCs [ 58 ].

A study using human umbilical cord Wharton's Jelly-derived MSCs (WS-MSCs) showed that Salvia miltiorrhiza increases the expression of nestin, β-tubulin, neurofilament, GFAP, and neurite outgrowth-promoting protein [ 59 ]. Another study in rat BM-MSCs showed that Salvia miltiorrhiza promotes Mash-1 and NGN-1 induced mRNA expression of TUJ-1, NF, and synaptophysin [ 60 ].

Ginkgolide B and Astragalus mongholicus can increase NSE-positive neuron-like cells and GFAP-positive astrocyte-like cells to promote MSCs differentiation into nerve cells [ 61 , 62 ]. Moreover, Astragalus mongholicus also enhances the expression of the Wnt-1 gene and Ngn-1 gene [ 62 ]. Ginsenoside Rg1 was found to accelerate the differentiation of neural phenotype in hASCs and upregulate NSE, MAP-2, GAP-43, NCAM, and SYN-1 genes [ 21 ]. Another study showed that gisenoside Rg1 can promote neural differentiation in mouse ASCs by miRNA-124 signalling pathway [ 63 ]. Similarly, radix Angelicae sinensis can also induce adipose-derived MSCs to differentiate into neuron-like cells [ 64 ]. In Ayurvedic medicine, dhanwantharam kashayam is considered a growth stimulant for children, which can promote nerve regeneration [ 65 ].

Angiogenesis effects

Treatment of hMSCs with olive leaf extract promoted the differentiation of cells into endothelial cells and development of the tubular construction needed for angiogenesis. At the same time, olive leaf extract can promote VEGF, PCAM, PDGF, and VEGFR-1 [ 7 ]. Curcumin is an antioxidant and anti-inflammatory substance in turmeric. Its ethanol extracts can increase the expression of CD34, CD133, and VEGFR2 to cause ASCs to proliferation and differentiation into endothelial progenitor cells [ 66 ]. In the animal hindlimb ischaemia model, fucoidan can protect MSCs from oxidative stress and enhance angiogenesis. Another study showed that fucoidan can inhibit the cell death caused by MSCs ischemia, and adjust the levels of apoptosis-related proteins and cellular ROS mainly by MnSOD and Akt pathways [ 67 ]. In addition, through ERK-IDO-1 signalling cascade, it increases the proliferation potential and the expression of cell cycle-associated proteins, and enhances the immunoregulation activity of MSCs [ 11 ]. Carica papaya leaf extract, rich in papain, was found to enhance the composition of IL-6 and stem cell factors related to platelet production in vitro [ 68 ].

Anti-adipogenic effects

Some plant extracts also have anti-adipogenic effects on MSCs. The results of one study confirmed that the antioxidant action of Tithonia diversifolia may influence the expression of HO-1. More importantly, it may regulate carbohydrate and fat metabolism by repressing adipocyte differentiation through activating AMPK [ 69 ]. In an experiment using MSCs, after stimulation with aloe-emodin, many indicators were reduced, including resistin, adiponectin, aP(2), lipoprotein lipase, PPARγ, and TNFα, which influence adipogenic pathways [ 70 ]. Quzhisu can repress adipogenic differentiation of BM-MSCs by downregulating PPARγ [ 71 ]. Similarly, flavonoids of epimedii like Quzhisu downregulate PPARγ, and can also decrease C/EBP-α [ 72 ].

Antioxidant stress effects

Undaria pinnatifida , Myrtus community L. and Cirsium setidens showed antioxidant stress effects in MSCs. Undaria pinnatifida , also called Mi-Yoek in Korea, is considered a healthy food. The anti-aging effect of Undaria pinnatifida in BM-MSCs was researched. The results showed that after H 2 O 2 treatment, it had the effect of antioxidant stress, and could decrease aging and improve the differentiation potential of cells by controlling ROS [ 73 ]. In addition, icariin protected rabbit BM-MSCs from oxygen, glucose, and apoptosis via inhibition of ERs-mediated autophagy associated with MAPK signalling [ 74 ]. Furthermore, residues from the production of Myrtus community L. can counteract the appearance of aging phenotypes in ASCs, reduce oxidative stress and inflammation, and enhance the expression of genes related to pluripotency [ 75 ]. The authors also studied the genetic programs responsible for cellular senescence in human ASCs exposed to oxidative stress and found that in the cells stimulated by Myrtle, the SA-β-Gal positive cells and the cell cycle regulation genes were decreased, while TERT and c-Myc genes were increased [ 76 ]. Cirsium setidens [ 77 ] has a suppressive effect on cell injury by regulating oxidative stress and repressing apoptosis-related signalling pathways.

The above studies have shown that certain plant extracts can promote the proliferation and differentiation of MSCs. However, some studies have demonstrated that plant extracts have side effects. Cimicifugae Rhizoma , also called Shengma in China, affects the vita of dental stem cells, and has side effects on the oral cavity at a high content [ 78 ]. Additionally, Asiasarum radix is the same as Cimicifugae Rhizoma [ 79 ]. As mentioned above, Fructus Ligustri Lucidi has an osteogenesis effect. Nevertheless, Fructus Ligustri Lucidi in a dose of more than 200 μg/mL has cytotoxicity to MSCs [ 27 ].

The effect of plant extracts on NSCs

Endogenous NSCs are abundantly in the subventricular region of the hippocampal granular area and the germinal area of the cerebrum. They differentiate cells according to the needs of brain structure and function [ 80 ]. NSCs are primarily used to remedy central nervous system injury and degenerative diseases. There are two intervention strategies involving NSCs. One strategy involves using endogenous NSCs to repair the diseased site, but endogenous NSCs are not sufficient and prefer to differentiate into gliocytes instead of neurons; the other strategy is transplantation of exogenous NSCs [ 61 ]. However, it is difficult to control the survival, replication, and differentiation of exogenous NSCs into local nerve cells. Sources of NSCs include human ESCs, human iPSCs, human foetal brain-derived neural stem/progenitor cells, and direct reprogramming of astrocytes. According to their functions, they can be divided into pluripotent and multipotent cell types [ 81 ]. Plant extracts play an important part in promoting the proliferation and differentiation of NSCs into new neurons. The Notch, Wnt, BMP, and sonic hedgehog signalling pathways have been the most studied [ 82 ].

Ginsenosides Rg1 advances the incorporation of Bromo-2-deoxyuridine and the expression of nestin and vimentin in NSCs, and promotes the proliferation of NSCs [ 83 ]. In addition, ginsenoside Rd can enhance the proliferation of NSCs in vivo and in vitro. It can enhance the size and quantity of neurospheres [ 84 ]. After oxygen and glucose deprivation (OGD) /r injury in vitro, resveratrol up-regulated the survival and proliferation of NSCs, and increased patched-1, smoothened (SMO) and Gli-1 [ 85 ]. Meanwhile, resveratrol can reduce the damage and raise the proliferation of NSCs by promoting Nrf2, HO-1 and NQO1 [ 86 ]. Artesunate is a derivative of artemisinin from Artemisia annua [ 87 ]. It can inhibit transcription by inducing Foxo-3a phosphorylation, then downregulating p27kip1, and enhancing the proliferation of NSCs in the infarcted cortex through PI3K/AKT signalling transduction [ 88 ].

Wnt/β-catenin pathway

The role of ginkgolide B has been mentioned above, it also can enhance the differentiation of NSCs after cerebral ischemia and may improve neural function by increasing the expression of BDNF, EGF, and SOCS2 [ 12 ]. Ginkgo biloba extract and Ginkgolide B, was found to accelerate cell cycle exit and neuronal differentiation in NSCs. Furthermore, ginkgolide B up-regulated the nuclear level of β-catenin and activated the classical Wnt to promote neuronal differentiation [ 89 ]. Curcumin ( Curcuma aromatica Salisb.) has some problems with pharmacokinetics and pharmacodynamics. Thereby Tiwari SK et al. prepared curcumin nanoparticles and found that they can activate the classic Wnt/β-catenin pathway to lead to human neurogenesis [ 90 ]. Icariin is an important biologically active ingredient extracted from Epimedium and has neuroprotective properties. Icariin treatment enhanced NSCs neurosphere formation and promoted the expression of nestin, β-III-tubulin and GFAP. Icariin-regulated genes participate in pathways including the Wnt and bFGF signalling [ 91 ], ERK/MAPK signalling [ 92 ], and BDNF-TrkB-ERK/Akt signalling pathway [ 93 ].

PI3K/AKT signalling pathway

One study evaluated the function of salvianolic acid B on the differentiation, proliferation, and neurite growth of mouse NSCs. The proper dose of salvianolic acid B promoted the quality of NSCs and neurospheres, and accelerates the growth of neurites of NSCs and their differentiation into neurons [ 94 ]. Zhuang P et al. selected 45 kinds of ingredients from TCM widely applied in the clinical treatment of stroke in China and examined their proliferation-inducing activity on NSCs. Finally, it was found that salvianolic acid B maintains NSCs self-renewal and promotes proliferation through the PI3K/Akt signalling pathway [ 95 ]. Salidroside is an ingredient extracted from the plant Rhodiola rosea L. It can inhibit hypoxic NSCs injury by increasing miR-210, thereby repressing BTG3 and influencing PI3K/AKT/mTOR signalling pathway [ 96 ]. The protective effect of berberine on OGD-treated cells via inhibiting the cell cycle. It can decrease cyclin D1, p53 and caspase 3, increase the phosphorylation level of p-Bad/tBad, and upregulate PI3K and Akt [ 97 ].

BMP signalling pathway

( +)-Cholesten-3-one( Serratula ) induced NSCs differentiation into dopaminergic neurons and promoted tyrosine hydroxylase, dopamine transporter, dopa decarboxylase, dopamine secretion, and evidently increased BMPR IB. The p-Smad1/5/8 expression indicates that ( +)-Cholesten-3-one may influence the BMP signalling [ 98 ].

Notch signalling pathway

Astragaloside IV is an ingredient in Astragalus membranaceus . Astragaloside IV leads NSCs to β-tubulin III ( +) and GFAP ( +) cells through the Notch signalling pathway [ 10 ]. Moreover, in an in vivo study, astragaloside IV can promote proliferative cells(BrdU + ), premature neurons (DCX + ), early proliferative cells (BrdU + /DCX + ), proliferative radial Glia-like cells (BrdU + /GFAP + ), and regulate the homeostasis of the CXCL1/CXCR2 signalling pathway [ 99 ].

Panax notoginseng saponins notably increased NSCs proliferation and the expression of nestin/BrdU, Tuj-1, and vimentin mRNA in hippocampal NSCs. And the results indicate that Panax notoginseng saponins may promote the proliferation and differentiation of NCSs after OGD in vitro by increasing the area density, optical density and the number of nestin/BrdU, nestin/vimentin, and nestin/tuj-1 positive cells [ 100 ]. One study investigated the effects of tetramethylpyrazine, an active element of Ligusticum Chuanxiong , which promotes the differentiation of NSCs into neurons, increases the phosphorylation of ERK1/2, and reduces the phosphorylation of p38 [ 101 ]. Baicalin could increase MAP-2 positive cells and decrease the number of GFAP stained cells. Meanwhile, p-STAT3 and Hes1 were downregulated, and NeuroD1 and Mash1 were upregulated. These results suggested that baicalin can promote neural differentiation but inhibit the formation of glial cells. Its role in promoting neurogenesis is related to STAT3 and bHLH genes [ 102 , 103 ]. Earlier research on NSCs showed that Buyanghuanwu decoction can promote cell growth and differentiation, increase neurofilament (NF) positive cells and GFAP positive cells, and promote intracellular Ca 2+ concentrations [ 104 , 105 ]. Jiaweisinisan has antidepressant effects, promotes hippocampal neurogenesis after stress damage, and significantly increases nestin, β-tubulin-III, and GFAP [ 106 ].

The effect of plant extracts on ESCs

ESCs can be obtained from the inner cell mass of a blastocyst. It has the characteristics of in vitro culture capacity, immortal cell proliferation, self-renewal, and multidirectional differentiation [ 107 ]. Using ESCs to differentiate into different cell models is a promising drug discovery method and technology [ 108 ]. Kami-Shoyo-San is a TCM that can protect neuronal apoptosis in ESCs by promoting brain-derived neurotrophic factor/tropomyosin receptor kinase B signalling pathway [ 109 ].

The effect of plant extracts on iPSCs

iPSCs have characteristics similar to those of ESCs in terms of unlimited self-renewal and differentiation capabilities. Plant extracts induce iPSCs production and apoptosis. The Sagunja-tang herbal formula can efficiently produce iPSCs from human foreskin fibroblasts via transcription factors [ 110 ]. Prunellae Spica and Magnoliae cortex -mediated apoptosis of undifferentiated iPSCs was found to be p53-dependent, and to have potent anti-teratoma activity, with no genotoxicity toward differentiated cells. Therefore, these compounds can be used for iPSC-based cell therapy to induce apoptosis of possible undifferentiated iPSCs and prevent the occurrence of teratomas [ 111 , 112 ].

Plant extracts can induce differentiation of iPSCs into nerve cells. Salvia miltiorrhiza can significantly increase the expression of nestin and microtubule-associated protein 2 (MAP2) genes and proteins, and induce the differentiation of iPSCs into neurons [ 113 ]. Plant extracts also have an improved effect on the nerve cell model differentiated from iPSCs. N-Butylidenephthalide ( n -BP) is derived from Angelica Sinensis . N -BP can reduce Aβ40 deposits, total tau protein, and its hyperphosphorylated form in iPSCs-derived neurons induced by Down syndrome [ 114 ]. Graptopetalum paraguayense can improve AD-related phenotypes, such as reducing Aβ 40, Aβ 42, and tau protein phosphorylation [ 115 ].

iPSCs are differentiated into cardiomyocytes, which are used in the research of related diseases. One study found that Salvia miltiorrhiza and Crataegus pentagyna have anti-arrhythmic effects. Salvia miltiorrhiza has an antioxidant effect, regulates calcium treatment on myocardial cells during I/R and decreases arrhythmia and apoptosis [ 116 ]. Crataegus pentagyna extract has an anti-arrhythmic effect on cardiomyocytes derived from human arrhythmia-specific iPSCs [ 117 ]. In addition, Yixinshu capsule has a protective effect on human iPSCs-derived cardiomyocytes by reducing endothelin 1 (ET-1) induced contractile dysfunction, increasing brain natriuretic peptide (BNP) content, and inducing morphological changes [ 118 ]. However, some plant extracts are toxic to cardiomyocytes, such as liensinine and neferine [ 119 ], mitragynine [ 120 ], and Erythrina senegalensis DC [ 121 ].

The effect of plant extracts on other stem cells

Ultraviolet-B (UVB) irradiation can damage the epidermis. Andrographis paniculata [ 122 ] promotes the proliferation of epidermal stem cells (EpSCs) and anti-aging via increasing integrin β1 and VEGF expression. Morin [ 123 ] and Vanillin [ 124 ] significantly inhibited UVB-induced damage to human keratinocyte stem cells, and effectively enriched the p53- specific ligasing ability of the mouse double minute 2 homologue in UVB irradiation-induced p53 activation. Likewise, zingerone ( Zingiber officinale Rosc.) [ 125 ] can protect the epidermis by restraining the UV damage mediated by p42/44 MAPK and p38 MAPK. In other stem cells, Ginkgo biloba [ 126 , 127 ] activates telomerase through PI3k/Akt signalling pathway to delay the aging of endothelial progenitor cells. Additionally, starting from telomerase, TSY-1 [ 128 ] increases telomerase activity in CD34 + haematopoietic stem cells.

Immune cell therapies

Adoptive cell therapy (ACT) is a kind of immunotherapy that is genetically modified T-cells to deliver a CAR or TCR. To a certain extent, mutated cancer cells provide many peptides that are not found in natural cells, which brings a potential target for constructing a new antigen screening system, and promotes the development of ACT, making CAR-T and TCR-T treatment become the most prospective way to treat cancer. However, ACT has great differences in the treatment of various tumours types, and there are still some shortcomings that need improvement [ 129 ].

CAR is a kind of engineering, which can enable lymphocytes to identify and eliminate cells delivering homologous target ligands. It has antigen binding domain, hinge, transmembrane domain and intracellular signal domain modules. By changing each component, its function and anti-tumour effect can be adjusted. At present, various types of CARs are being developed and designed to improve the safety and effectiveness in cancer treatment [ 130 ]. Clinically, treatment with CAR-T-cells first requires T-cells, which can be obtained from the patient’s peripheral blood, or allogeneic CAR-T-cells obtained from a donor [ 131 ]. T-cells are stimulated and expanded in vitro and transduced with specific CAR genes through viral vectors, and then, the CAR-T-cells are infused back into the patient to perform the set tumour-killing effect in the patient's body. This type of therapy is also called CAR-T-cell therapy (Fig.  3 ).

CAR-T-cell therapy and the four generations of improvements. The first-generation CARs were fused with a single-chain variable fragment (scFv) to a transmembrane domain and an intracellular signalling unit: the CD3 zeta chain. Then, the second-generation CARs improved the costimulatory molecule receptor-like CD28, which is the most commonly used. The second-generation CARs increased the production of cytokines and enhanced durability. The third-generation of CARs design in-corporated an additional costimulatory domain to enhance CAR function and included the scFv, the initial CD3ζ-chain, and the CD28 and 4-1BB or OX40 costimulatory domains [ 132 ]. At present, fourth-generation CAR-T therapy has been extended. In this type of CAR-T-cell therapy, cytokine genes have been added to the structure, which can stimulate high cytokines expression that enhances the activity of T-cells after CAR-T-cells are activated, thereby improving the antitumour activity of CAR-T-cells [ 133 ]

In 2017, the FDA approved two types anti-CD19 CAR-T-cell products to treat both B-cell ALL and diffuse large B-cell lymphoma, and these products have transformed the field of anticancer immunotherapy [ 134 ]. However, there are still some limitations in CAR-T-cell therapy. Mechanisms hampering CAR-T-cell efficiency include limited T-cell persistence and therapy-related toxicity. Furthermore, severe toxicities, restricted trafficking to, infiltration into and activation within tumours, antigen escape and heterogeneity; manufacturing issues; physical properties; and the immunosuppressive capacities of solid tumours have prevented the success of CAR-T-cells in these entities [ 135 , 136 ]. Additionally, it may not be possible to obtain a sufficient number of T-cells from the patient because the patient is usually not considered for CAR-T-cell therapy, usually due to a reduction in the number of original lymphocytes caused by previous cytotoxic treatment [ 134 ]. In application, almost all CAR-T-cell products are derived from CD4 + T-cells and CD8 + T-cells, both cell populations likely contribute to treatment effect [ 134 ]. Some plant extracts have beneficial effects on CD4 + T-cells and CD8 + T-cells. For example, Fuzheng Qingjie [ 137 , 138 ], Fuzheng Fangai [ 139 ], Xiaoji [ 13 ], Cistanche deserticola [ 140 ], Epimedium koreanum Nakai [ 141 ], Glycyrrhiza uralensis [ 142 , 143 ], Aidi [ 144 ], and Scolopendra subspinipes [ 145 , 146 , 147 ] can increase in CD4 + cells and the CD4/CD8 ratio, and produce FN-g, IL-2, IL-4, IL-6, and IL-7; Xiao Ai Ping [ 148 ], Lycium barbarum [ 149 , 150 , 151 ], Dangguibuxue tang [ 152 ], Oldenlandia diffusa [ 153 , 154 , 155 ], Carthamus tinctorius [ 156 ], lectin-55 [ 157 ], and Tricosanthes kirilow [ 158 ] have an effect on increasing in CD8 + cells, and tumour infiltration and increasing IFN-g and IL-10. Shenqi Fuzheng [ 159 ], Lycium barbarum [ 160 ], Ganoderma lucidum [ 161 ], Yunzhi-Danshen [ 162 ] can upregulate CD3 + , CD4 + , CD4 + /CD8 + and NK + cells. Moreover, gastrodin was found to ameliorated the CD8 + T-cell-mediated immune response and significantly improved protection in tumour-challenged animals. This finding indicates that gastrodin is a potential adjuvant contributing to anticancer immunomodulation.

On the other hand, the tumour microenvironment is a complex pathological system composed of tumour cells, blood/lymphatic vessels, tumour stroma, and tumour-infiltrating myeloid precursors, providing a living environment for tumour cells and promoting tumour metastasis. In the tumour microenvironment, tumour-infiltrating myeloid precursors mainly include tumour-associated macrophages, tumour-associated dendritic cells, and myeloid-derived suppressor cells, which inhibit T-cells or other immune cells and play an important role in its antitumour activity. Therefore, improving the tumour microenvironment by targeting these cells is an effective way to assist CAR-T-cell therapy. Liu J et al. reviewed Chinese herbal medicine and its components that induce tumour cell apoptosis and directly inhibit tumour growth and invasion, providing new research ideas for cell therapy [ 163 ].

Due to the limitations of CAR in the application, it only recognizes cell surface protein antigens, while TCR can distinguish intracellular proteins expressed as peptides on MHC class I molecules. Therefore, TCR-T therapy has superiority in the field of solid tumour treatment. The TCR can be produced in two ways. One method is to identify and clone T- cells from patients with antitumour reactions. Their TCRs are inserted into retroviruses or lentiviruses to infect target T-cells. Another method is to isolate TCRs from humanized mice that recognize tumour antigens. TCRs can be immunized with appropriate tumour antigens because they can express human MHC class I or II. After T-cells were isolated, the TCR gene was cloned into a recombinant vector for genetic engineering transformation of patients' autologous T-cells [ 164 ].

Although effective responses have been observed in TCR-T-cell therapy, adverse reactions have become a thorny issue in many trials. Most of the reasons are that TCR-T- cells, in addition to their killing effect on tumour cells, severely destroy normal cells with the same antigen [ 129 ]. Since TCR-T-cells have only emerged in recent years, there are almost no plant extracts currently used in TCR-T-cell research. Parvifoline AA is an ent-kaurane diterpenoid and can significantly stimulate the level of NKG2D ligands on hepatocellular carcinoma cells, evidently enhancing their recognition and lysis by NK cells [ 14 ]. Perhaps improving the efficacy of TCR-T-cells in the immunosuppressive microenvironment and determining that the expression is mainly (if not completely) limited to cancer cell targets may be a future research direction for plant extracts.

Plant extracts are relatively easy to obtain and have significant activity in the treatment of many diseases. The above review shows that plant extracts have an effect on stem cell proliferation or directed differentiation and play an important role in solving the problem of insufficient endogenous stem cells and directed differentiation of stem cells; In immune cell therapy, the effect of plant extracts on stem cells are reflected in the beneficial effects on CD4 + T-cells and CD8 + T-cells and the improvement of the tumour microenvironment. Moreover, plant extracts, such as astragaloside [ 165 ], paeoniflorin [ 166 ], and licorice [ 167 ], have a good immunoregulatory and anti-inflammatory activities and may provide a better treatment plan for the cytokine storm caused by cell therapy [ 168 ]. At present, cell therapy is promising. However, to understand the long-term effects, more in-depth research on the dose and side effects of plant extract applications is still needed. Although plant extracts are recognized as excellent alternatives to synthetic interventions, clinical application is challenging due to the variability and complexity of the bioactive components present in the extracts, as well as the effects of solvents during extraction. Therefore, the effects of plant extracts on cell therapy need to be better and more deeply researched to supplement the current deficiencies in cell therapy.

Availability of data and materials

Not applicable.

Abbreviations

Mesenchymal stem cells

Nerve stem cells

Chimeric antigen receptor T-cell immunotherapy

T-cell receptor modified T-cell immunotherapy

Adipose-derived stem cells

Induced pluripotent stem cells

Embryonic stem cells

Human leukocyte antigen-antigen D related

Bone marrow-derived human MSCs

Extracellular signal-regulated kinase

Runt-related transcription factor 2

Hutchinson–Gilford progeria syndrome

Vascular endothelial growth factor

Vascular endothelium growth factor receptor

Traditional Chinese medicine

Alkaline phosphatase

Peroxisome proliferator activated receptor γ

BuShenNingXin decoction

Wharton's jelly-derived MSCs

CCAAT enhancer-binding protein-α

Oxygen and glucose deprivation

Basic fibroblast growth factor

Neural stem/progenitor cells

Glial fibrillary acidic protein

Butylidenephthalide

Endothelin 1

Brain natriuretic peptide

Ultraviolet-B

Epidermal stem cells

Adoptive cell therapy

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Acknowledgements

We thank Lei Tong for guidance in drawing from University of science and technology Beijing.

This study was supported by fundamental Research Funds for the Central public welfare research institutes of China (ZZ13-YQ-082-C1; JBGS2021001), the Scientific and Technological Innovation Project of China Academy of Chinese Medical Sciences (CI2021A00610).

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H.J Yang, X.Y. Li and P. Chen designed the idea of this review; C.F. Li, Zh. Cui, S.W. Deng and P. Chen co-wrote the paper with input from all authors. All authors read and approved the final manuscript.

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Additional file 1: table s1..

Plant Extracts for the Osteogenesis of MSCs.

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Li, C., Cui, Z., Deng, S. et al. The potential of plant extracts in cell therapy. Stem Cell Res Ther 13 , 472 (2022). https://doi.org/10.1186/s13287-022-03152-z

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April 16, 2024

Rejuvenating the immune system by depleting certain stem cells

At a glance.

• Researchers found that depleting certain stem cells improved the immune systems of aged mice.
• The findings suggest that a similar treatment might be used to help protect older people against infections.

The risk for serious infections rises with age, as people’s immune systems lose the ability to respond to novel infections. Part of the reason for this is that the types of hematopoietic stem cells (HSCs), which make the various types of blood cells, change with age.

Some HSCs, called myeloid-biased HSCs (my-HSCs) produce mostly myeloid cells, which include red blood cells, platelets, and most cells of the innate immune system. Others, called balanced HSCs (bal-HSCs), produce a healthy mix of myeloid and lymphoid cells, which include the T and B cells that make up the adaptive immune system.

The proportion of my-HSCs increases with age. This leads to more myeloid cells and fewer lymphoid cells. More myeloid cells increase inflammation and bring an increased risk of atherosclerosis and myeloid-related diseases such as leukemia. Fewer lymphoid cells reduces the ability to fight infections. A research team led by Drs. Kim Hasenkrug and Lara Myers at NIH and Drs. Irving Weissman and Jason Ross at Stanford University School of Medicine explored whether reducing my-HSCs could restore a more “youthful” immune system in aged mice. The results appeared in Nature on March 27, 2024.

The team began by identifying proteins on the surface of mouse HSCs that are unique to my-HSCs. They then created antibodies against these proteins and used them to deplete my-HSCs in aged mice.

Depleting my-HSCs reduced the effects of aging on the mouse immune system. It increased lymphoid progenitor cells, which give rise to T and B cells, in the bone marrow. Consequently, treated mice had more naïve T cells and B cells in their blood than untreated mice. These cells allow the immune system to learn to recognize novel infections. The treatment also lowered levels of exhausted T cells and age-associated B cells, along with certain inflammatory markers. 

When the researchers vaccinated aged mice with a live, weakened virus, those with depleted my-HSCs had a stronger T cell response than untreated mice. The treated mice also gained better protection against infection from the vaccination.

These findings could explain why older people are more vulnerable to infections such as SARS-CoV-2. Weakened adaptive immunity from fewer lymphoid cells makes it harder for them to fight off the infection. At the same time, increased myeloid cells cause harmful inflammation. The researchers noted that the genes that characterize my-HSCs in mice are also found in aged human HSCs. This suggests that my-HSC depletion might be used in humans to relieve certain age-associated health problems.

“During the start of the COVID-19 pandemic, it quickly became clear that older people were dying in larger numbers than younger people,” Weissman says. “This trend continued even after vaccinations became available. If we can revitalize the aging human immune system like we did in mice, it could be lifesaving when the next global pathogen arises.”

—by Brian Doctrow, Ph.D.

Related Links

• Human Skeletal Stem Cell Identified
• Gene Therapy Reduces Need for Transfusions in Severe Blood Disorder
• Stem Cell Transplant Induces Multiple Sclerosis Remission
• Immune System Reset May Halt Multiple Sclerosis Progression
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References:  Depleting myeloid-biased haematopoietic stem cells rejuvenates aged immunity. Ross JB, Myers LM, Noh JJ, Collins MM, Carmody AB, Messer RJ, Dhuey E, Hasenkrug KJ, Weissman IL. Nature . 2024 Apr;628(8006):162-170. doi: 10.1038/s41586-024-07238-x. Epub 2024 Mar 27. PMID: 38538791.

Funding:  NIH’s National Institute of Allergy and Infectious Diseases (NIAID), National Cancer Institute (NCI), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), and National Institute of General Medical Sciences (NIGMS); Virginia and D.K. Ludwig Fund for Cancer Research; Stanford University; Radiological Society of North America; Stanford Cancer Institute.

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Replacing Animal Testing with Stem Cell-Organoids : Advantages and Limitations

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• Published: 19 April 2024

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• Guiyoung Park 1 ,
• Yeri Alice Rim 2 , 3 , 4 ,
• Yeowon Sohn 5 ,
• Yoojun Nam   ORCID: orcid.org/0000-0003-4583-3455 5 , 6 &
• Ji Hyeon Ju 2 , 3 , 4 , 6  

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Various groups including animal protection organizations, medical organizations, research centers, and even federal agencies such as the U.S. Food and Drug Administration, are working to minimize animal use in scientific experiments. This movement primarily stems from animal welfare and ethical concerns. However, recent advances in technology and new studies in medicine have contributed to an increase in animal experiments throughout the years. With the rapid increase in animal testing, concerns arise including ethical issues, high cost, complex procedures, and potential inaccuracies.

Alternative solutions have recently been investigated to address the problems of animal testing. Some of these technologies are related to stem cell technologies, such as organ-on-a-chip, organoids, and induced pluripotent stem cell models. The aim of the review is to focus on stem cell related methodologies, such as organoids, that can serve as an alternative to animal testing and discuss its advantages and limitations, alongside regulatory considerations.

Although stem cell related methodologies has shortcomings, it has potential to replace animal testing. Achieving this requires further research on stem cells, with potential societal and technological benefits.

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Use of Large Animal Models for Regenerative Medicine

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Introduction

Historically, animal models have contributed substantially to the advancement and study of vaccines, surgical techniques, and various scientific experiments [ 1 ]. However, owing to the problems associated with animal testing, researchers are now questioning whether animal models and tests are the best options for these procedures. Growing animal testing is ethically concerning amid scientific evolution. According to the Humane Society International Organization, more than 100 million animals are killed annually worldwide for scientific purposes (Humane Society International). The animals used vary depending on their traits and include rats, mice, rabbits, dogs, cats, guinea pigs, zebrafish, swine [ 2 , 3 ].

In December 2022, the U.S. Food and Drug Administration (FDA) announced animal testing is no longer mandatory safety approval of products [ 4 ]. However, products that are used on the human body still require safety testing. In other words, testing for toxicity, compatibility, and safety is compulsory for products; however, animal testing is unnecessary for conducting these tests. In response, research facilities and companies have introduced alternatives such as computer simulations and in silico models. Stem cell therapy has gained popularity throughout the medical field, and various studies are underway to gain deeper knowledge [ 5 ]. With the emergence of this stem cell-based test, alternative methods have also arisen, potentially offering to become a replacement for animal testing.

When comparing test options, alternatives offer more beneficial attributes than animal testing. Non-animal tests are cost-effective, less time-consuming, and simpler procedures than animal tests [ 6 ]. However, most research institutions use animal models. This is because animal testing has been a longstanding experimental approach for decades [ 7 , 8 ]. Efforts are being made to replace animal testing with the use of human cells, as animal testing results often exhibit interspecies differences with humans, thus lacking the ability to reliably predict clinical outcomes. Application of advancing stem cell technology continue, but completely replacing animal experimentation poses significant challenges. Therefore, it is important to conduct further studies to advance the science of alternative testing methods. This review aimed to summarize the use of stem cell technology as an alternative to animal testing and discuss its advantages and limitations.

Current State of Animal Testing

Uses of animal testing.

Animal testing has been used for decades, and in the 21st century, the number of tests has increased considerably [ 2 ]. With approximately 100 million animals used for testing annually worldwide, science has been rapidly evolving. The primary function of animal testing is to test drugs, their toxicity, and their compatibility with the human body to ensure safe use. Hence, pre-launch testing is crucial. Companies and research facilities must subject their products to clinical trials before introducing them to potential customers.

Neurological disorder such as Parkinson’s and Alzheimer’s have also been modeled in animals to understand their mechanisms and to determine suitable treatments [ 9 , 10 , 11 ]. For instance, in the case of Parkinson’s disease, various animal models have been employed, including Caenorhabditis elegans, Zebrafish, and mice. Additionally, genetically modified mice carrying mutations associated with proteins like α-synuclein, Parkin, Pink1, and LRRK2, as well as mice induced with α-Synuclein Pre-Formed Fibril (PFF), are utilized to assess dopaminergic neuronal loss and investigate changes in α-synuclein aggregation. In Alzheimer’s disease, transgenic mice carrying mutations associated with familial Alzheimer’s disease (FAD), such as the 5xFAD model, are commonly used. These models allow for the evaluation of amyloid beta reduction through histological methods and the assessment of drug efficacy using behavioral tests like the Maze, providing insights into underlying disease mechanisms. Animals utilized as disease models contribute significantly to our comprehensive understanding of the mechanisms behind various illnesses, facilitating our grasp of these conditions. Research conducted using these animal disease models has indeed contributed to the discovery and development of treatments. However, it’s scientifically crucial to acknowledge that these animal models often present disparities in lifespans compared to humans and may not entirely mirror the intricate etiology of human diseases. Additionally, while animal experimentation is utilized for various conditions such as cancer, diabetes mellitus, and traumatic brain injury, it’s constrained by its inability to fully capture the nuances of the human immune system and intricate disease mechanisms (Table  1 ).

In addition to modeling diseases, animals are also used to test cosmetics or healing rates of products. In the cosmetics industry, animals are typically used to test skin or eye irritation to assess the safety of these products in humans [ 17 , 18 ]. The Draize test, developed in 1944 to test for such hazards in rabbits [ 19 ], is used to test products such as drugs and balms for wound healing. It involves creating wounds on animals to gauge recovery rates [ 16 ].

Related laws, Guidelines, and Principles

As of 2023, current regulations state that the FDA no longer deems animal tests necessary for evaluating product safety [ 4 ]. This enables companies and research facilities to explore possible non-animal testing when obtaining product approval. Additionally, out of 195 countries worldwide, only 42 have laws or regulations limiting animal testing for products (The Humane Society). Animal testing laws have been implemented by banning animal testing or limiting its use during testing. Europe completely banned cosmetics tested on animal testing in 2013 [ 3 , 20 , 21 ]. This demonstrates a push to limit animal testing; however, the movement remains ineffective because of the absence of laws against animal testing in most countries.

Guidelines for animal experimentation and clinical trials for drug development and safety testing have varied procedures among companies and researchers up to now. So, the Guidance for Industry for Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals from the Center for Drug Evaluation and Research provides guidelines for the safety assessment of products compiled from regulatory standards of several countries. According to these guidelines, preclinical trial researchers should consider factors such as animal species, age, delivery method (dosage, administration, treatment regimen, etc.), and test material stability [ 22 ] (Fig. 1 ).

( A ) Procedure of new drug approval as stated by the Food and Drug Administration (FDA). In the preclinical research stage, small, medium, and large animals are usually used for testing new drugs. ( B ) iPSCs that can replacing animal testing. PBMCs or fibroblasts are reprogrammed to iPSCs and subsequently differentiated into target modeling cells such as neurons, cardiomyocytes, and hepatocytes. ( C ) iPSC-derived 3D organoids enable in vitro efficacy and safety testing. Organ-on-a-chip embedded with organoids used in in vitro tests, created using BioRender

The FDA has also provided a drug development process that includes these steps. The first step in drug development is discovering and researching a new drug (discovery and development stage). The second stage is preclinical research, in which drugs have to undergo a series of animal tests (or alternative tests, if possible) for safety. The FDA strongly suggests that animal preclinical trials follow Good Laboratory Practice (GLP). The main elements of GLP are as follows [ 23 ]: appropriate use of qualified personnel, quality assurance, appropriate use of facility and care for animals, proper operating procedures for animals used in trial, individual animal data collection and evaluation, testing product properly handled and analyzed, study proceeds with an approved protocol, data should be collected as outlined in the protocol, and full report prepared after procedures.

To enhance clinical translation, reproducibility issues in preclinical trials, such as biased allocation, insufficient controls, and lack of interdisciplinary, uncharacterized, or poorly characterized supplies [ 24 ]. The third step involves clinical testing on humans to assess safety and efficacy. The fourth and fifth stages comprise FDA post-market safety monitoring for all approved drugs [ 25 ].

Guidelines also suggest the 3R (replacement, reduction, and refinement) principle, which recommends that scientists follow certain criteria during clinical trials. Replacement involves using other testing methods other than animal testing [ 26 ]. In computer models, tissues, or stem cell research, if alternatives to animal testing exist, researchers should prioritize their use. Reduction involves minimizing the number of animal tests [ 26 ]. Questioning the necessity of animal tests during a particular part of our research and reducing their numbers imbues the concept with meaning. Refinement focuses on minimizing stress and providing the best care to animals [ 26 ], including providing proper food, entertainment, and clean well-maintained shelters.

As International efforts for animal replacement methods, research and development into alternative testing methods is already underway in both Europe and the United States, with each regulatory body establishing its own initiatives. In Europe, the European Center for the Validation of Alternative Methods (ECVAM) was founded in 1992, and since 2013, the sale of cosmetics containing ingredients tested on animals has been completely banned. Moreover, there are plans to expand the scope to include medical devices, health supplements, and pharmaceuticals in the future. In the United States, the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) was established in 2000. The objective is to reduce animal testing by 2025 and eliminate mammalian animal testing entirely by 2035 through innovative advancements in alternative testing methodologies. In 2022, amendments to the Food, Drug, and Cosmetic Act in the United States removed mandatory animal testing requirements in the drug development stage and presented alternative testing methods as viable non-clinical trial options.

Problems/limitations of Animal Testing

A pressing issue with animal testing is the ethical concerns stemming from it. Most studies have demonstrated that these models undergo invasive procedures that often result in pain or even death. Research indicates that animals share pain and emotional capacity with humans [ 27 ]. Thus, sacrificing them for research can appear cruel. Advocates call for equitable treatment, opposing animal testing as inhumane and cruel. Such ethical issues has always followed animal testing and are ongoing [ 28 ].

Moreover, some studies have indicated that animal testing is not an accurate model for medicines or substances, highlighting the need for accurate and efficient testing alternatives that are similar humans. The complexity of human disease mechanisms raises doubts whether animal models can accurately replicate them.

Physiological differences between animals and humans mean a product safe for animals may not guarantee human safety [ 29 ]. Interspecies differences have led to poor results in correlating animal testing with human outcomes, consequently causing several clinical trial failures [ 30 ]. Between 2010 and 2017, clinical trials for drugs had a greater chance of failing phase І, owing to safety and efficacy [ 31 ]. In addition, even if a product passes phase І there is still a 90% rate of failure while undergoing the necessary procedures [ 32 , 33 ]. Prolonged use of animal testing can ultimately endanger humans, as some drugs and products approved through trials were later deemed harmful. Concerns such as high cost and long laborious procedures will be discussed below.

Benefits of Replacing Animal Testing

The main benefits of replacing animal tests with alternatives are as follows: cost-effective, time efficient, less complex testing procedures, and societal benefits.

Stem cell modeling is less expensive than animal testing. The Draize test mentioned before costs approximately $1,800, whereas non-animal testing methods cost considerably less [ 6 ]. Affordable procedures offer renewed chances for past costly research to emerge. A decrease in the cost of procedures would facilitate new drug development, making opportunities for new technologies easier.

Animal testing requires prior preparation that is often complex and time consuming. Several guidelines of various organizations worldwide follow certain principles and procedures. For animal testing, factors such as providing clean and well-maintained shelters, food, necessary supplies for survival, and entertainment are laborious [ 26 ]. Alternatives are time-efficient and less laborious, simpler protocols, and fewer supplies to maintain procedures.

Alternatives to Animal Testing Related to Stem Cells

Organoids are organ-like structures derived from self-organizing stem cells in 3D cell cultures. They exhibit organ-specific characteristics and originate from stem cells undergoing self-organization [ 34 , 35 ]. . They are beneficial over previous 2D cell culture, as they can show near-physiological cellular composition and actions [ 36 ]. Organoids are typically established from embryonic stem cells (ESCs), human pluripotent stem cells (PSCs), and adult stem cells [ 37 , 38 , 39 ]. The potential of organoids as alternatives stems from their correlation with patient reactions to products such as drugs, indicating that they are a promising for rare diseases where clinical trials are impractical [ 39 ]. Organoids have a wide range of applications and are suitable for studies of infectious diseases, hereditary diseases, and toxicity, and can provide personalized medicine for individual patients [ 38 ].

Recent studies have shown that PSC organoids can form complex brain organoids that are useful for modeling traumatic brain injury [ 15 ]. Organoids derived from PSCs are of various types, including stomach, lung, liver, kidney, cerebral, and thyroid, and can contribute to organ failure or dysfunction. Cancer organoids are cultured from thin tumor sections, which are efficient for studying cancer syndromes [ 34 ]. Organoid studies on Alzheimer’s disease highlight the possibility of using familial or sporadic Alzheimer’s disease induced pluripotent stem cells (iPSCs) to model brain activity [ 40 ]. Thyroid follicles derived from hESCs have the potential to be used as organoids to treat hypothyroidism [ 41 ] (Table  2 ). Technology development of 3D bioprinting organoids is underway, promising better productivity. Bioprinting for organoids includes inkjet-based bioprinting, laser-assisted bioprinting, extrusion-based bioprinting, and photo-curing bioprinting [ 42 ]. Ongoing studies are also exploring 3D printing technology using organoids, offering the possibility of creating organs for patient-tailored services and toxicology research.

However, organoids still possess limitations that render them unsuitable tools to replace animal testing. Organoids lack of vasculature structure affects growth and maturation, leading to differences in behavior compared to the original tissue [ 59 ]. This may result in only partial replication, leading to an incomplete disease model [ 38 ]. Moreover, the complexity and heterogeneity of certain organs, such as the brain or immune system, pose challenges for complete replication in organoid models. This inability to replicate such complexity can affect the translatability of findings from organoid studies to clinical applications. Research and experiments involving organoids often require lengthy culture protocols, which can vary depending on the type of organoid being cultivated. In some extreme cases, organoid culture may extend for months or even years, as seen in examples such as intestinal organoids(8 weeks or more), retinal organoids(6 ~ 39 weeks or more), brain organoids(12 weeks or more), and liver organoids(4 ~ 8 weeks or more) [ 60 , 61 , 62 , 63 , 64 ]. Even after going through the lengthy process, there are sometimes a lack of established organoids in sufficient numbers. This limited availability of organoids can hinder the procedure of functional testing, which can lead to insufficient research outcomes. Organoids also lack the intricate network of connections that can be seen in living organisms. Inter-organ communication is crucial when checking metabolic health, and with organoids lacking such an important factor, it is difficult to create treatments for any abnormalities regarding infection and diseases. Organoids also lack a diverse set of cell types, structural organization, and physiological functions in comparison to functioning organs, which limits the ability to accurately replicate disease processes and responses to treatment [ 59 ]. When compared to animal models, organoids fall behind, as animal models offer a broader view of processes for diseases, immune responses, and systemic effects of treatments. Another noteworthy concern arises from the fact that current production technology for organoids under GMP (Good Manufacturing Practice) standards has yet to be established.

Quality Control of Organoid

For organoids to serve as suitable models for diseases or experimental purposes, quality control (QC) is essential. Accuracy and consistency in production lead to more precise results, ensuring better therapeutic treatments or modeling. If quality control for organoids isn’t established sufficiently, problems such as inconsistent test results, misinterpretation of existing data, wastage of valuable resources, reproducibility issues, unreliable models, and ethical concerns regarding biomedical studies could arise.

Organoid structures and functions can be assessed through multiple methods. Structural assessment of organoids can be performed using bright-field imaging for both quantitative and qualitative research. Additionally, methods such as immunofluorescent staining, transmission electron microscopy, and scanning electron microscopy are also utilized [ 65 , 66 ]. The functionality of organoids can be assessed through qPCR and single-cell or bulk cell RNA sequencing, which provide quantitation of marker gene expression, revealing cell identity and composition [ 67 ]. Assay methods like ELISA and colorimetric assays are useful for secretome quantification while Luciferase essays help measure enzyme activity [ 65 , 68 ]. Staining methods such as Glycosaminoglycan (GAG) staining(specifically for synovial mesenchymal stromal cell (SMSC) organoids), immunofluorescence staining, and Alizarin red staining mainly help with visualizing components within the organoid [ 65 , 68 , 69 ]. There are also more direct methods like implantation to test the in vivo functions of organoids [ 65 , 70 ] (Table  3 ).

Extracellular microenvironment, which contain such things as soluble bioactive molecules, extracellular matrix, and biofluid flow, contributes to the growth rate and formation of organoids. Given the variation in extracellular microenvironments across different types of organoids, it is imperative to modulate the extracellular microenvironment accordingly for each organoid type. This ensures the production of organoids with consistent quality across different production batches [ 71 ].

Regulations/Applications Regarding Organoids from the FDA

While there aren’t any specific regulations regarding organoids from the FDA(Food and Drug Administrations) as of in the recent years, there are two categories of applications that include framework for cell related therapies, which include organoids. There are two applications, Biologics License Application (BLA) and the Investigational New Drug (IND) Application. The BLA, as stated in the official website of FDA, is a request for permission to introduce and deliver for a biologic product(vaccines, somatic cells, gene therapy, tissues, recombinant therapeutic proteins, organoids, etc.) into interstate commerce. Requirements for a BLA includes applicant information, product/manufacturing information, pre-clinical studies, clinical studies, and labeling. The IND application is a request for authorization to administer an investigation drug or biological product to humans. IND had three types: Investigator IND, Emergency Use IND, and Treatment IND which could fall into two categories being commercial or non-commercial. The IND application must contain the following broad areas of information: Animal Pharmacology and Toxicology studies, Manufacturing Information, Clinical protocols and Investigator Information.

When examining the current ongoing clinical trials( ClinicalTrials.gov ) in the application of organoids, it can be noted that they are being utilized in refractory cancers, osteosarcoma, high-grade glioma, advanced breast cancer, and colorectal cancer. This pertains to the utilization of the organoid platform to investigate the sensitivity to various drugs (chemotherapy, hormonal therapy, targeted therapy) by exposing them to each individual agent (or combination of agents). It is anticipated and ongoing to aid in clinical decisions regarding the optimal treatment option for each patient.

Organ-on-a-chip

Organoid chips(OoC) can be regarded as the outcome of merging biology and microtechnology, serving as microfluidic cell culture devices [ 72 , 73 ]. OoC has the ability to mimic the cellular environment, which leads to an examination of their effects on cell communication with more accessibility and ease. The chips are generally designed by collecting cells (primary cells, transformed cell lines, human ESC, or iPSCs) using equipment with pumps(that enable fluid flow), incubators, sensors, and microscopes to monitor and examine the cells in the system [ 49 , 74 ] (Fig.  1 ). Depending on the type or cell or method cells can be aggregated in matrix or matrixless conditions [ 75 ].

Various types of human organ chips, including the liver, heart, eyes, kidneys, bones, intestines, and skin, are used to simulate the breathing motion. Single-organ chips such as liver-on-a-chip and lung-on-a-chip are useful for observing individual chemical reactions [ 53 ]. There are also multiple organ-on-chip, which are organ-chips connected to a vast system [ 76 ]. The main purpose of multi-organ-on-chips is to simulate the entire body, recognizing that a single organ does not represent the entire human system. Using multiple organ-on-chips connected to one system allows the analysis of how various organs communicate with each other.

The U.S. Food and Drug Administration (FDA) and the U.S. National Institutes of Health (NIH) have provided project support for tissue chips for drug screening, including lung-on-a-chip. Additionally, efforts are being made globally to advance the utilization of organoid chips, such as the establishment of the European Organ-on-Chip Society in Europe.

A limitation of OoCs is their complex experimental setup [ 77 ], which can be avoided with clear guidelines or protocols. Cell medium changes also raise concerns about chip environments [ 77 ]. There is also the issue of using animal models to validate OoC systems initially [ 78 ]. To address this, OoC experts recommend forming well-established collaborations with developers, toxicologists, and pharmaceutical companies to explore alternative solutions.

iPSCs(Induced Pluripotent stem Cells)

iPSCs are a recent development in the field of disease modeling. Having traits such as self-renewal and pluripotency, iPSCs can transform into various cells within the human body (Fig.  1 ); thus, reprogramming patient cells creates personalized medicine for specific diseases [ 79 , 80 ]. The ability to produce a large batch of iPSCs with only a small number of patient samples is important [ 81 , 82 ]. The objectives of iPSC models closely align with the 3R principle [ 83 ]. Replacing animal models in research while adhering to reduction and refinement principles is expected to be advantageous.

iPSCs are research to find cures for various diseases and are used as broad disease models (Table  2 ). For example, iPSCs from patients with Parkinson’s disease differentiate into midbrain dopaminergic neurons (DAns) in the substantia nigra pars compacta (SNpc), which can be used to model Parkinson’s disease on a cellular basis [ 43 , 44 , 45 ]. For cardiac diseases, which include a decrease in cardiomyocytes that leads to scar formation and ultimately heart function failure, there are existing studies that explore iPSCs for novel therapeutic cures [ 84 ]. iPSC-derived progenitors such as human HCN4 + and human ESC derived ROR2+, CD13+, KDR+, PDGFRα + cells later generate cardiomyocytes [ 47 ]. For cancer modeling using iPSCs, reprogrammed tumor specimens or iPSCs with premalignant or early genetic lesions can show the stages of cancer [ 49 ]. iPSCs from patients that are healthy and those with Alzheimer’s disease differentiate into the main brain cells, modeling the human brain with a functional blood barrier. Further research could drive drug discovery [ 9 ]. Studies of organ failure or dysfunction have shown that human iPSCs are useful. Research on lung regeneration has shown that endogenous and exogenous stem cells mediate therapeutic results [ 50 ]. Another study focused on the use of liver hepatoblasts, which could help alleviate hepatotoxicity through liver development and hepatic differentiation [ 85 ].

However, iPSCs are still in a relatively early developmental phase and have several limitations. Concerns for researchers regarding iPSCs is in vitro culture adaptation and tumorigenicity, the inability to completely reflect in vivo 3D environments, and the variation of differentiated cells depending on the protocol [ 86 , 87 ]. Quality control of differentiated cells and influencing factors are crucial for iPSC researchers, impacting their applicability as medical models or treatments.

Figure 2 Human diagram showing multiple stem cell-related technologies that can be applied to various human organs.

A BioRender diagram depicts diverse stem cell technologies for human organs

Limitations

Stem cell-related methodologies, such as organoids, are a very new technology in the field of animal alternative testing. In the early developmental stage, alternative stem cell models and technologies still require a few years of testing. Animal testing is still used today, owing to its historical role in safety and efficacy assessment. New alternatives have been presented; however, the uncertainty of these methods have caused most researchers to adhere to old protocols. In cases of complex diseases arising from various factors such as cardiovascular, neurodegenerative, and infertility, complete replacement by animal alternative testing methods may still be impractical. In such instances, it is crucial to concurrently employ animal experimentation alongside alternative testing methods utilizing organoids or stem cells to bolster data reliability. As a component of these endeavors, numerous researchers have undertaken disease modeling, such as stroke, utilizing brain organoids and cardiac organoids in in vitro experiments. The solution involves focusing on alternative testing methods [ 88 ]. By transforming old methods and creating alternatives, this shift could be the norm. There has already been a move toward that goal, as the FDA has established a cross-agency working group (The Alternative Methods Working Group) to promote various alternative methods, such as in vivo, in vitro, in silico , or system toxicology modeling [ 89 ]. In the 2021, FDA report titled “Advancing Regulatory Science at FDA,” the most prioritized area is identified as “Advancing Novel Technologies to Improve Predictivity of Non-clinical Studies and Replace, Reduce, and Refine Reliance on Animal Testing.”

Given ongoing research in alternative stem cell-related methods, this appears promising to replace animal testing. These alternatives offer advantages for scientists and the public. However, it is important to acknowledge that iPSCs, organoids, and OoCs each have distinct strengths and limitations. With continued advancements and studies to further understand these issues, these limitations can be avoided.

Data Availability

All data pertaining to this manuscript are included within the article.

Abbreviations

Food and Drug Administration

organ-on-chip

induced pluripotent stem cell

pluripotent stem cell

Embryonic stem cell

Center for Drug Evaluation and Research, GLP, Good Laboratory Practice

Dopaminergic neurons

Substantia Nigra pars compacta

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This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number : HI22C1314).

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Park, G., Rim, Y.A., Sohn, Y. et al. Replacing Animal Testing with Stem Cell-Organoids : Advantages and Limitations. Stem Cell Rev and Rep (2024). https://doi.org/10.1007/s12015-024-10723-5

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• Orthop Rev (Pavia)
• v.14(3); 2022

Stem cells: a comprehensive review of origins and emerging clinical roles in medical practice

Salomon poliwoda.

1 Department of Anesthesiology, Mount Sinai Medical Center

2 LSU Health Science Center Shreveport School of Medicine, Shreveport, LA

Amanda Schaaf

3 University of Arizona College of Medicine-Phoenix, Phoenix, AZ

Abigail Cantwell

Latha ganti.

4 Department of Emergency Medicine, University of Central Florida

Alan D. Kaye

5 Department of Anesthesiology, Louisiana State University Health Sciences Center Shreveport

Luke I. Mosel

Caroline b. carroll, omar viswanath.

6 Department of Anesthesiology, Louisiana State University Health Sciences Center Shreveport, Innovative Pain and Wellness, Creighton University School of Medicine

Stem cells are types of cells that have unique ability to self-renew and to differentiate into more than one cell lineage. They are considered building blocks of tissues and organs. Over recent decades, they have been studied and utilized for repair and regenerative medicine. One way to classify these cells is based on their differentiation capacity. Totipotent stem cells can give rise to any cell of an embryo but also to extra-embryonic tissue as well. Pluripotent stem cells are limited to any of the three embryonic germ layers; however, they cannot differentiate into extra-embryonic tissue. Multipotent stem cells can only differentiate into one germ line tissue. Oligopotent and unipotent stem cells are seen in adult organ tissues that have committed to a cell lineage. Another way to differentiate these cells is based on their origins. Stem cells can be extracted from different sources, including bone marrow, amniotic cells, adipose tissue, umbilical cord, and placental tissue. Stem cells began their role in modern regenerative medicine in the 1950’s with the first bone marrow transplantation occurring in 1956. Stem cell therapies are at present indicated for a range of clinical conditions beyond traditional origins to treat genetic blood diseases and have seen substantial success. In this regard, emerging use for stem cells is their potential to treat pain states and neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease. Stem cells offer hope in neurodegeneration to replace neurons damaged during certain disease states. This review compares stem cells arising from these different sources of origin and include clinical roles for stem cells in modern medical practice.

I. Introduction

Stem cells are a unique population of cells present in all stages of life that possess the ability to self-renew and differentiate into multiple cell lineages. These cells are key mediators in the development of neonates and in restorative processes after injury or disease as they are the source from which specific cell types within differentiated tissues and organs are derived. 1 Within the neonate stage of life stem cells serve to differentiate and proliferate into the multitude of cell types and lineages required for continuing development, while in adults their primary role is regenerative and restorative in nature. 2 Stem cells have unique properties that set them apart from terminally differentiated cells allowing for their specific physiological roles. The ability of stem cells to differentiate into multiple cell types is termed potency, and stem cells can be classified by their potential for differentiation as well as by their origin. Totipotent or omnipotent stem cells can form embryonic tissues and can differentiate into all cell lineages required for an adult. Pluripotent stem cells can differentiate into all three germ layers while multipotent stem cells may only differentiate into one kind of germ line tissue. Oligopotent and unipotent stem cells are the type seen in adult organ tissues that have committed to a cell lineage and can only diversify into cell types within that lineage. 1 Embryonic stem cells are derived from the inner cell mass of a blastocysts and are totipotent. The range of their use is typically restricted due to legal and ethical factors and for this reason mesenchymal stem cells are typically preferred. Mesenchymal stem cells can be isolated from a variety of both neonate and adult tissues but still maintain the ability to differentiate into multiple cell types allowing for their clinical and research utilization without the ethical issues associated with embryonic stem cells. 3

Another key feature of stem cells is their ability to self-renew and proliferate providing a continuous supply of progeny to replace aging or damaged cells. During the developmental phase this proliferation allows for the growth necessary to mature into an adult. After the developmental phase has concluded, this continued proliferation allows for healing and restoration on a cellular level after tissue or organ injury has taken place. 2 These physiological and developmental characteristics make stem cells an integral part in the field of regenerative medicine due to their ability to generate entire tissues and organs from just a handful of progenitor cells.

Stem cells began their role in modern regenerative medicine in the 1950’s with the first bone marrow transplantation occurring in 1956. This breakthrough shed light on the potential treatments possible in the future with further development and refinement of clinical techniques and paved the way for the stem cell therapies that are now available. 4,5 Stem cell therapies are now indicated for a range of clinical conditions beyond traditional origins to treat genetic blood diseases and have seen substantial success where other treatments have fallen short. One emerging use for stem cells is their potential to treat paint states and neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease. Stem cells offer the hope in the setting of neurodegeneration to replace the neurons damaged during the pathogenesis of certain diseases, a goal not achievable utilizing current technologies and methods. 6

Organ bioengineering is yet another a rapidly developing and exciting new application for stem cells with both clinical and research implications. 7 Immunosuppression free organ transplants are now a possibility with the advancement organ manufacturing utilizing the patient’s own cells. 8 This along with the potential for eliminating organ donor waiting lists is an enticing prospect, but many technological developments are necessary before this technology can be implemented in clinical settings on a wide scale. Research has already benefitted greatly from this field because organ like tissues can be grown in lab settings to model disease progression. This offers the potential to develop new treatments while determining their efficacy on a cellular level without risk to patients. 9,10

Currently one of the most prolific clinical uses of stem cells in the field of regenerative medicine is to treat inherited blood diseases. Within these diseases a genetic defect or defects prevents the proper function of cells derived from the hematopoietic stem cell lineage. Treatment includes implantation of genetically normal cells from a healthy donor to serve as a lifelong self-renewing source of normally functioning blood cells. However these treatments are limited by the availability of suitable donors. 11

Stem cells can be derived from multiple sources including adult tissues or neonatal tissues such as the umbilical cord or placenta. Embryonic stem cells have been utilized in the past for research, but ethical concerns have led to them being replaced largely by stem cells derived from other origins. 12 Common tissues from which adult oligopotent and unipotent stem cells are isolated include bone marrow, adipose tissue, and trabecular bone. 13 Bone marrow has traditionally been the most common site from which to extract non neonatal derived stem cells but involves an invasive and painful procedure. Peripheral blood progenitor cells have been utilized to avoid harvesting cells from bone marrow. However, this technique has issues and risks of its own and was initially a less potent source of stem cells. It is also now known that stem cells differ in their proliferative and differentiation potential based on their origin. Cells sourced from umbilical Wharton’s jelly and adipose tissue were found to proliferate significantly more quickly than cells sourced from bone marrow and placental sources. 14,15

A rapidly advancing source of stem cells known as induced pluripotent stem cells (iPSC’s) are now being utilized clinically as well. These iPSC’s are derived from somatic cells that have been reprogrammed back to a pluripotent state utilizing reprogramming factors and require less invasive techniques to harvest in comparison to traditional sources. 16,17 Once returned to a pluripotent state, the cells then undergo a process called directed differentiation in which they are converted into desired cell types. Directed differentiation is achieved by mimicking microenvironments and extracellular signals in vitro in a manner that produces predictable cell types. 18 In the future, this technique could provide a novel form of personalized gene therapy in which oligopotent or unipotent cells are procured from tissue, reprogrammed back to a less differentiated state, and then reintroduced into a different location within the patient. Work is also being done to combine this technique with modern gene editing methods to provide an entirely new subset of therapies. 19 This method of transplantation would greatly reduce the chance for rejection and does not require a suitable donor, as the cells are sourced from the patient being treated. 20,21

II. Bone marrow as a source for stem cells

Stem cells are required by self-renewing tissues to replace damaged and aging cells because of normal biological processes. Both myeloid and lymphoid lineage cells derived from hematopoietic stem cells are relatively short-lived cell types and require a continuous source of newly differentiated replacement cells. 22 Hematopoietic stem cells (HSC’s) are those that reside within the bone marrow and provide a source for the multiple types of blood cells required for normal physiological and immunological functions. These cells inhabit a physiological niche which allows them to undergo the process of asymmetric division. When stem cells divide asymmetrically the progeny of the division includes one identical daughter cell but also results in the production of a differentiated daughter cell. Differentiation of these daughter cell into specialized cell types is guided by certain microenvironments, extrinsic cues, and growth factors that the cell comes in contact with. 23,24 This mechanism allows for bone marrow stem cell numbers to stay relatively constant despite sustained proliferation and differentiation of progeny taking place. 22,25,26

HSC’s are the most studied class of adult tissue derived stem cells and their clinical potential was recognized early in the history of regenerative medicine. At the beginning of the 1960’s, HSC’s were isolated from bone marrow and therapeutic models in mice induced with leukemia were developed in order to show the efficacy of bone marrow derived stem cell treatments. Success in these experiments led to further refinement of techniques and by the 1970’s and 80’s clinical stem cell transplants were a regular occurrence and began to make the impact on blood diseases that we continue to see today. 27,28

Bone marrow has historically been the predominant harvesting site for stem cell collection due to its accessibility, early identification as a source, and lengthy research history. Isolating stem cell from bone marrow involves an invasive and painful surgical procedure and does come with a risk hospitalization or other complications. Patients also report increased post procedural pain and pre-procedural anxiety when compared with other harvesting techniques. 29,30 Bone marrow however has proved to be a denser source of cells than other harvesting methods yielding 18 times more cells than peripheral blood progenitor cell harvesting techniques initially. As technology and methods improved however, it was found that treating patients with a cytokine treatment prior to peripheral blood progenitor cell harvesting mobilized many of the desired cells into the blood stream and drastically increased the efficacy of this technique, making it clinically viable. 31–33 In a double blinded randomized study 40 patients underwent bone marrow and peripheral blood progenitor cell collections and the yield of useable harvested cells were compared. It was found that blood progenitor cell collection yielded significantly more useable stem cells and patients were able to undergo the collection procedure more frequently when compared to the bone marrow harvesting method. 32 This, coupled with the invasiveness and risks associated with harvesting stem cells from bone marrow have increased peripheral blood progenitor cell collections popularity.

Overall, bone marrow as a reservoir of stem cells continues to be a clinical and research necessity related to its well understood and documented history as a source of viable stem cells and track record of efficacy. According to the European Group for Blood and Marrow Transplantation, only one fatal event was recorded stemming from the first 27,770 hematopoietic stem cell transplants sourced from bone marrow during the period of 1993-2005. 34 This undeniable track record of safety coupled with clinicians’ experience performing bone marrow transplant procedures guarantees the continued use of bone marrow as a source of HSC’s for the near future.

III. Amniotic cells as a source for stem cells

Historically, the two most common types of pluripotent stem cells include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). 35 However, despite the many research efforts to improve ESC and iPSC technologies, there are still enormous clinical challenges. 35 Two significant issues posed by ESC and iPSC technologies include low survival rate of transplanted cells and tumorigenicity. 35 Recently, researchers have isolated pluripotent stem cells from gestational tissues such as amniotic fluid and the placental membrane. 35 Human amnion-derived stem cells (hADSCs), including amniotic epithelial cells and amniotic mesenchymal cells, are a relatively new stem cell source that have been found to have several advantageous characteristics. 35,36

For background, human amniotic stem cells begin emerging during the second week of gestation when a small cavity forms within the blastocyst and primordial cells lining this cavity are differentiated into amnioblasts. 36 Human amniotic epithelial stem cells (hAESCs) are formed when epiblasts differentiate into amnioblasts, whereas human amniotic mesenchymal stem cells (hAMSCs) are formed when hypoblasts differentiate into amnioblasts. 35,36 This differentiation occurs prior to gastrulation, so amnioblasts do not belong to one of the 3 germ layers, making them theoretically pluripotent. 35–37

Previously, pluripotency and immunomodulation are qualities that have been thought to be mutually exclusive, as pluripotency has traditionally been regarded as a characteristic limited to embryonic stem cells whereas immunomodulation has been a recognized property of mesenchymal stem cells. 36 However, many recent studies have found that these two qualities coexist in hADSCs. 35,36

In recent years, hADSCs, including human amniotic epithelial stem cells (hAESCs) and human amniotic mesenchymal stem cells (hAMSCs) have been attractive cell sources for clinical trials and medical research, and have been shown to have advantages over other stem cells types. 35,37 These advantages include low immunogenicity and high histocompatibility, no tumorigenicity, immunomodulatory effects, and significant paracrine effects. 35 Also, several studies have evaluated the proangiogenic ability of hADSCs. 35 Interestingly, they found that hAMSCs were shown to augment blood perfusion and capillary architecture when transplanted into ischemic limbs of mice, suggesting that hAMSCs stimulate neovascularization. 35,38 Additionally, another advantage is that hADSCs are easier to obtain compared to other stem cell sources, such as bone marrow stem cells (BMSCs). 35

Regarding the low immunogenicity, hADSCs have been shown to have a low expression of major histocompatibility class I antigen ( HLA-ABC ), and no expression of major histocompatibility class II antigen ( HLA-DR ), β2 microglobulin, and HLA-ABC costimulatory molecules, including CD40, CD80 and CD8635. Notably, there have been reports of transplantation of hAMSCs into patients with lysosomal diseases who had no obvious rejection. 35 Moreover, a recent study demonstrated no hemolysis, allergic reactions, or tumor formations in mice who received intravenous hAESCs. 35,39

Additionally, studies have demonstrated that both hAESCs and hAMSCs have great potential to play an important role in regenerative medicine. They both have demonstrated that they can differentiate into several specialized cells, including adipocytes, bone cells, nerve cells, cardiomyocytes, skeletal muscle cells, hepatocytes, hematopoietic cells, endothelial cells, kidney cells, and retinal cells. 35

Multiple preclinical studies have revealed the potential for hADSCs to be used in the treatment of several diseases including premature ovarian failure, diabetes mellitus, inflammatory bowel disease, brain/spine diseases, and more. 35,40,41 For example, one preclinical study investigated the effect of hAMSC-therapy on ovarian function in natural aging ovaries within mice. 40 They found that after the hAMSCs were transplanted into the mice, the hAMSCs significantly improved follicle proliferation and therefore ovarian function. 40 Another study investigated the effect of hAESC-therapy on outcomes after stroke in mice. 41 They found that, administration of hAESCs after acute (within 1.5 hours) stroke in mice reduced brain infarct development, inflammation, and functional deficits. 41 Additionally, they found that after late administration (1-3 days poststroke) of hAESCs, functional recovery in the mice was still improved. 41 Overall, they concluded that administration of hAESCs following a stroke in mice showed a significant neuroprotective effect and facilitated repair and recovery of the brain. 41

Although a number of preclinical studies, like the ones previously described, have shown considerable promise regarding the use of ADSC-therapy, more studies are needed. Future studies can continue to work toward determining if hADSCs are capable of being used for cell replacement and better elucidate the mechanisms by which hADSCs work.

IV. Adipose tissue as a source for stem cells

Although the use of bone marrow stem cells (BMSCs) is now standard, dilemmas regarding harvesting techniques and the potential for low cell yields has driven researchers to search for other mesenchymal stem cell (MSCs) sources. 42 One source that has been investigated is human adipose tissue. 42

After enzymatic digestion of adipose tissue, a heterogenous group of adipocyte precursors are generated within a group of cells called the stromal vascular fraction (SVF). 42 Adipose-derived stem cells (ADSCs) are found in the SVF. 42,43 Studies have demonstrated that ADSCs possess properties typically associated with MSCs, and that they have been found to express several CD markers that MSCs characteristically express. 43 ADSCs are multipotent and have been shown to differentiate into other cells of mesodermal origin, including osteoblasts, chondroblasts, myocytes, tendocytes, and more, upon in vitro induction. 42–45 Additionally, ADSCs have demonstrated in vitro capacity for multi-lineage differentiation into specialized cells, like insulin-secreting cells. 43,46

A significant advantage of ADSCs over BMSCs is how easy they are to harvest. 43,45 White adipose tissue (WAT) contains an abundance of ADSCs. 43 The main stores of WAT in humans are subcutaneous stores in the buttocks, thighs, abdomen and visceral depots. 43 Due to this, ADSCs can be harvested relatively easily by liposuction procedures from these areas of the body. 43,45 Moreover, ADSCs make up as much as 1-2% of the SVF within WAT, sometimes even nearing 30% in some tissues. 43,45 This is a significant difference from the .0001-.0002% stem cells present in bone marrow. 43 Given this difference in stem cell concentration between the sources, there will be more ADSCs per sample of WAT compared to stem cells per bone marrow sample, further demonstrating an easier acquisition of stem cells when using adipose tissue.

Another advantage of ADSCs is their immune privilege status due to a lack of major histocompatibility complex II (MHC II) and costimulatory molecules. 42,43,45,47 Some studies have even demonstrated a higher immunosuppression capacity in ADSCs compared to BMSCs as ADSCs expressed lower levels of human antigen class I (HLA I) antigen. 47 They also have a unique secretome and can produce immunomodulatory, anti-apoptotic, hematopoietic, and angiogenic factors that can help with repair of tissues – characteristics that may support successful transplantations without the need for immunosuppression. 42–45 Moreover, ADSCs have the ability to be reprogrammed to induced pluripotent stem (iPS) cells. 43

The number of ADSC clinical trials has risen over the past decade, and some have shown significant promise. They have demonstrated abilities to differentiate into multiple cell lines in a reproducible manner and be safe for both autogenetic and allogeneic transplantations. 45 Several recent studies have demonstrated that ADSC-therapy may potentially be useful in the treatment of several conditions, including diabetes mellitus, Crohn’s disease, multiple sclerosis, fistulas, arthritis, ischemic pathologies, cardiac injury, spinal injury, bone injuries and more. 44–48

One clinical trial conducted in 2013 investigated the therapeutic effect of co-infusion of autologous adipose-derived differentiated insulin-secreting stem cells and hematopoietic stem cells (HSCs) on patients with insulin-dependent diabetes mellitus. 46 Ten patients were followed over an average of about thirty-two months, and they found that all the patients had improvement in C-peptide, HbA1c, blood sugar status, and exogenous insulin requirement. 46 Notably, there were no unpleasant side effects of the treatment and all ten patients had rehabilitated to a normal, unrestricted diet and lifestyle. 46

In another 4-patient clinical trial in which ADSCs were used to heal fistulas in patients with Crohn’s disease, full healing occurred in 6 out of the 8 fistulas with partial healing in the remaining two. 44 No complications were observed in the patients 12 months following the trial. 44 Although these results are promising, the mechanism by which the healing took place remains unclear. When considering the properties of ADSCs, there are a number of factors that could have played a role in the healing, such as the result of paracrine expression of angiogenic and/or anti-apoptotic factors, stem cell differentiation, and/or local immunosuppression. 44

Other exciting studies have demonstrated a use of ADSCs in the treatment of osteoarthritis (OA). One meta-analysis compared the use of ADSCs and BMSCs in the treatment of osteoarthritis. 47 This meta-analysis included 14 studies comprising 461 original patient records. 47 Overall, the comparison between treatment of OA didn’t show a significant difference in the disease severity score change rate between patients treated with ADSCs and those treated with BMSCs. 47 However, there was significantly more variability in the outcomes of those treated with BMSCs with the highest change rate being 79.65% in one study and the lowest being 22.57% in another study. 47 Given this, ADSCs may represent a more stable cell source for the treatment of OA. 47 Although this study is specific to OA treatment, it is worth acknowledging the possibility that ADSCs may also represent a more stable cell source for treatment of other diseases as well.

Though recent ADSC research, as described above, has been promising, unfortunately reproducible in vivo studies are still lacking in both quality and quantity. 42 Therefore, further studies are necessary prior to progression to routine patient administration. 42

V. Umbilical Cord as a source for stem cells

Umbilical Cord stem cells can be drawn from a variety of locations including umbilical cord blood, umbilical cord perivascular cells, umbilical vein endothelial cells, umbilical lining, chorion, and amnion. Umbilical cord blood can be drawn with minimal risk to the donor, and it has been used since 1988 as a source for hematopoietic stem cells. 49 When compared to stem cells obtained from bone marrow, umbilical cord derived stem cells are much more readily available. With a birth rate of more than a 100 million people per year globally, there is a lot of opportunity to use umbilical cord blood as a source for stem cells.

The process of extracting the blood is very simple and involves a venipuncture followed by drainage into a sterile anti-coagulant-filled blood bag. It is then cryopreserved and stored in liquid nitrogen. There are quite a few benefits to utilizing umbilical cord stem cells rather than stem cells drawn from adults. One of the biggest benefits is that the cells are more immature which means that there is a lower chance of rejection after implantation in a host and would lead to decreased rates of graft-versus-host disease. They also can differentiate into a very wide variety of tissues. For example, when compared with bone marrow stem cells or mobilized peripheral blood, umbilical cord blood stem cells have a greater repopulating ability. 50 Cord blood derived CD34+ cells have very potent hematopoietic abilities, and this is attributed to the immaturity of the stem cells relative to adult derived cells. Studies have been done that analyze long term survival of children with hematologic disorders who were transplanted with umbilical cord blood from a sibling donor. These studied revealed the same or better survival in the children that received the umbilical cord blood relative to those that got transplantation from bone marrow cells. Furthermore, rates of relapse were the same for both umbilical cord blood and bone marrow transplant. 51

One of the unique features of stem cells taken from umbilical cord blood is the potential to differentiate into a wide variety of cell types. There are three different kinds of stem cells that can be found in the umbilical cord blood which include hematopoietic, mesenchymal, and embryonic-like stem cells. Not only can these cell types all renew themselves, but they can differentiate into many different mature cell types through a complex number of signaling pathways. This means that these cells could give rise to not only hematopoietic cells but bone, neural and endothelial cells. There are studies taking place currently to see if umbilical cord blood derived stem cells can be utilized for cardiomyogenic purposes. Several studies have showed the ability to transform umbilical cord blood mesenchymal stem cells into cells of cardiomyogenic lineage utilizing activations of Wnt signaling pathways. 52 Studies are also being conducted on the potential of neurological applications. If successful, this could help diseases such as cerebral palsy, stroke, spinal cord injury and neurodegenerative diseases. Given these cell’s ability to differentiate into tissues from the mesoderm, endoderm and ectoderm, they could be utilized for neurological issues in place of embryonic stem cells that are currently extremely controversial. 53 There are currently studies involving in vitro work, pre-clinical animal studies, and patient clinical trials, all for the application of stem cells in neurological applications. There is big potential for the use of umbilical blood stem cells in the future of regenerative medicine.

VI. Placental Tissue as a Source for Stem Cells

Placental tissue contains both stem cells and epithelial cells that can differentiate into a wide variety of tissue types which include adipogenic, myogenic, hepatogenic, osteogenic, cardiac, endothelial, pancreatic, pulmonary, and neurological. Placental cells can differentiate in to all these different kinds of tissues due to lineages originating from different parts of the placenta such as the hematopoietic cells that come from the chorion, allantois, and yolk sac while the mesenchymal lineages come from the chorion and the amnion. 54 It can be helpful to think of human fetal placental cells as being divided into four different groups: amniotic epithelial cells, amniotic mesenchymal stromal cells, chorionic mesenchymal stromal cells and chorionic trophoblast cells. 54

Human amniotic epithelial cells (hAECs) can be obtained from the amnion membrane where they are then enzymatically digested to be separated from the chorion. When cultured under certain settings hAECs have been found to be able to produce neuronal cells that synthesize acetylcholine, norepinephrine as well as dopamine. 55,56 This ability would mean they have potential for regenerative purposes in diseases such as Parkinson’s Disease, multiple sclerosis, and spinal cord injury. There is also research being done to utilize hAECs for ophthalmological purposes, lung fibrosis, liver disease, metabolic diseases, and familial hypercholesterolemia. Once cultured, hAECs have been shown to produce both albumin and alpha-fetoprotein as well as showing ability to store glycogen. Furthermore, they have been found to metabolize ammonia and testosterone. In more recent studies conducted in mouse models, these cells have been found to have therapeutic efficacy after transplantation for cirrhosis. 57

Mesenchymal stem cells are in many different tissues such as the bone marrow, umbilical cord blood, adipose tissue, Wharton’s jelly, amniotic fluid, lungs, muscle and the placenta. Placental mesenchymal stromal cells specifically originate from the extraembryonic mesoderm. Human amniotic mesenchymal stromal cells (hAMSCs) and chorionic mesenchymal stromal cells (hCMSCs) have both been found to have very low levels of HLA-A,B,C. This means that they have immune privileged profiles for potential transplantation. 58,59 Placental derived mesenchymal stem cells have been shown to have expression of CD29, CD44, CD105 and CD166 which is the same as adipose derived mesenchymal stem cells. These markers have been shown to have osteogenic differentiating abilities. 57 An interesting element of placental mesenchymal stem cells is that their properties differ depending on the gestational age of the placenta. When cells are harvested at lower gestational ages, they show faster generation doubling times, better proliferative abilities, wider differentiation potential and more phenotypic stability than cells harvested from placental tissue that is considered to be at term. 60 Furthermore, they have great potential to be used clinically. Placental mesenchymal stromal cells have been studied for use in treating acute graft-versus-host disease that was refractory to steroid treatment. Studies have shown that the 1-year survival rates in patients treated with placenta derived stromal cells were 73% while retrospective control only showed 6% survival. 61 Placenta derived MSCs have also been found to aid in wound healing and could potentially be used to aid with certain inherited skin conditions such as epidermolysis bullosa. 62

Stem cells are diverse in their differentiation capacity as well as their source of origin. As we can see from this review, there are similarities and differences when these cells are extracted from different sources. Research has shown initial promise in neurodegenerative diseases such as Alzheimer’s and Parkinson’s Disease. It has also shown to be beneficial in the areas of musculoskeletal regenerative medicine and other pain states. Organ bioengineering for transplantation is another potential benefit that stem cells may offer. For these reasons, extensive research is still needed in this area of medicine to pave the way for new developing therapy modalities.

Conflict of Interest of each author

Dedications.

This review is dedicated to Dr. Justine C. Goldberg MD

Funding Statement

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• 10 April 2024

How to supercharge cancer-fighting cells: give them stem-cell skills

• Sara Reardon 0

Sara Reardon is a freelance journalist based in Bozeman, Montana.

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A CAR T cell (orange; artificially coloured) attacks a cancer cell (green). Credit: Eye Of Science/SPL

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Bioengineered immune cells have been shown to attack and even cure cancer , but they tend to get exhausted if the fight goes on for a long time. Now, two separate research teams have found a way to rejuvenate these cells: make them more like stem cells .

Both teams found that the bespoke immune cells called CAR T cells gain new vigour if engineered to have high levels of a particular protein. These boosted CAR T cells have gene activity similar to that of stem cells and a renewed ability to fend off cancer . Both papers were published today in Nature 1 , 2 .

The papers “open a new avenue for engineering therapeutic T cells for cancer patients”, says Tuoqi Wu, an immunologist at the University of Texas Southwestern in Dallas who was not involved in the research.

Reviving exhausted cells

CAR T cells are made from the immune cells called T cells, which are isolated from the blood of person who is going to receive treatment for cancer or another disease. The cells are genetically modified to recognize and attack specific proteins — called chimeric antigen receptors (CARs) — on the surface of disease-causing cells and reinfused into the person being treated.

But keeping the cells active for long enough to eliminate cancer has proved challenging, especially in solid tumours such as those of the breast and lung. (CAR T cells have been more effective in treating leukaemia and other blood cancers.) So scientists are searching for better ways to help CAR T cells to multiply more quickly and last longer in the body.

Cutting-edge CAR-T cancer therapy is now made in India — at one-tenth the cost

With this goal in mind, a team led by immunologist Crystal Mackall at Stanford University in California and cell and gene therapy researcher Evan Weber at the University of Pennsylvania in Philadelphia compared samples of CAR T cells used to treat people with leukaemia 1 . In some of the recipients, the cancer had responded well to treatment; in others, it had not.

The researchers analysed the role of cellular proteins that regulate gene activity and serve as master switches in the T cells. They found a set of 41 genes that were more active in the CAR T cells associated with a good response to treatment than in cells associated with a poor response. All 41 genes seemed to be regulated by a master-switch protein called FOXO1.

The researchers then altered CAR T cells to make them produce more FOXO1 than usual. Gene activity in these cells began to look like that of T memory stem cells, which recognize cancer and respond to it quickly.

The researchers then injected the engineered cells into mice with various types of cancer. Extra FOXO1 made the CAR T cells better at reducing both solid tumours and blood cancers. The stem-cell-like cells shrank a mouse’s tumour more completely and lasted longer in the body than did standard CAR T cells.

Master-switch molecule

A separate team led by immunologists Phillip Darcy, Junyun Lai and Paul Beavis at Peter MacCallum Cancer Centre in Melbourne, Australia, reached the same conclusion with different methods 2 . Their team was examining the effect of IL-15, an immune-signalling molecule that is administered alongside CAR T cells in some clinical trials. IL-15 helps to switch T cells to a stem-like state, but the cells can get stuck there instead of maturing to fight cancer.

The team analysed gene activity in CAR T cells and found that IL-15 turned on genes associated with FOXO1. The researchers engineered CAR T cells to produce extra-high levels of FOXO1 and showed that they became more stem-like, but also reached maturity and fought cancer without becoming exhausted. “It’s the ideal situation,” Darcy says.

Stem-cell and genetic therapies make a healthy marriage

The team also found that extra-high levels of FOXO1 improved the CAR T cells’ metabolism, allowing them to last much longer when infused into mice. “We were surprised by the magnitude of the effect,” says Beavis.

Mackall says she was excited to see that FOXO1 worked the same way in mice and humans. “It means this is pretty fundamental,” she says.

Engineering CAR T cells that overexpress FOXO1 might be fairly simple to test in people with cancer, although Mackall says researchers will need to determine which people and types of cancer are most likely to respond well to rejuvenated cells. Darcy says that his team is already speaking to clinical researchers about testing FOXO1 in CAR T cells — trials that could start within two years.

And Weber points to an ongoing clinical trial in which people with leukaemia are receiving CAR T cells genetically engineered to produce unusually high levels of another master-switch protein called c-Jun, which also helps T cells avoid exhaustion. The trial’s results have not been released yet, but Mackall says she suspects the same system could be applied to FOXO1 and that overexpressing both proteins might make the cells even more powerful.

Nature 628 , 486 (2024)

doi: https://doi.org/10.1038/d41586-024-01043-2

Doan, A. et al. Nature https://doi.org/10.1038/s41586-024-07300-8 (2024).

Article   Google Scholar  

Chan, J. D. et al. Nature https://doi.org/10.1038/s41586-024-07242-1 (2024).

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