stem cell research studies

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Stem-cell research articles from across Nature Portfolio

Stem-cell research is the area of research that studies the properties of stem cells and their potential use in medicine. As stem cells are the source of all tissues, understanding their properties helps in our understanding of the healthy and diseased body's development and homeostasis.

Latest Research and Reviews

Genome engineering with Cas9 and AAV repair templates generates frequent concatemeric insertions of viral vectors

AAV vectors form difficult-to-detect concatemers at Cas9 target sites.

• Fabian P. Suchy
• Daiki Karigane
• Hiromitsu Nakauchi

A two-way street – cellular metabolism and myofibroblast contraction

• Birgit Sawitzki
• Georg N. Duda

Comparison studies identify mesenchymal stromal cells with potent regenerative activity in osteoarthritis treatment

• Hongshang Chu
• Shaoyang Zhang

ATG or post-transplant cyclophosphamide to prevent GVHD in matched unrelated stem cell transplantation?

• Olaf Penack
• Mouad Abouqateb
• Zinaida Peric

Depleting myeloid-biased haematopoietic stem cells rejuvenates aged immunity

Antibody-mediated depletion of myeloid-biased haematopoietic stem cells in aged mice restores characteristic features of a more youthful immune system.

• Jason B. Ross
• Lara M. Myers
• Irving L. Weissman

Generating human bone marrow organoids for disease modeling and drug discovery

This protocol can be used to generate three-dimensional vascularized bone marrow organoids from human induced pluripotent stem cells. The organoids contain key stromal and hematopoietic cell types and can be engrafted with normal and malignant cells from adult donors to model niche interactions.

• Aude-Anais Olijnik
• Antonio Rodriguez-Romera
• Abdullah O. Khan

News and Comment

Improving the EASIX’ predictive power for NRM in adults undergoing allogeneic hematopoietic cell transplantation

• Silvia Escribano-Serrat
• Luis Gerardo Rodríguez-Lobato
• María Queralt Salas

Minimally invasive derivation of primary human epithelial organoids from fetal fluids

Primary fetal organoids are currently derived from tissue samples obtained at termination of pregnancy. We developed an approach that enables prenatal derivation of epithelial organoids from fetal fluids. Single-cell mapping of the human amniotic fluid content unveiled the presence of viable fetal epithelial progenitors of multiple tissues that can form fetal lung, kidney and intestinal organoids.

How to decrease bone marrow collection volume and risk contaminations via the operating room cell concentration?

• Yoann Grimaud
• Flore Sicre de Fontbrune
• Lionel Faivre

Early lymphocyte reconstitution and viral infections in adolescents and adults transplanted for sickle cell disease

• Loïc Vasseur
• Alexis Cuffel
• Nathalie Dhédin

Post-transplant cyclophosphamide with Sirolimus or Cyclosporine for GvHD prophylaxis in matched related and unrelated transplantation: a two-center analysis on 213 consecutive patients

• Simona Piemontese
• Maria Teresa Lupo Stanghellini
• Patrizia Chiusolo

Donor NKG2D rs1049174 polymorphism predicts hematopoietic recovery and event-free survival after single-unit cord blood transplantation in adults

• Takaaki Konuma
• Megumi Hamatani-Asakura
• Satoshi Takahashi

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For decades, researchers have been trying to expand human blood stem cells in culture. Researchers at the institute have recently accomplished this, opening the way to explore many new medical therapies and avenues of basic research.

Omair Khan named Soros Fellow

Stem Cell MD/PhD Student Omair Khan became one of 23 graduate students nationally to be awarded a 2023 Paul and Daisy Soros Fellowship for New Americans.

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Stem Cell Therapy: a Look at Current Research, Regulations, and Remaining Hurdles

Stem cell therapies offer great promise for a wide range of diseases and conditions. However, stem cell research—particularly human embryonic stem cell research—has also been a source of ongoing ethical, religious, and political controversy.

In September 2014, the Sanford Stem Cell Clinical Center at the University of California, San Diego (UCSD) Health System announced the launch of a groundbreaking clinical trial to assess the safety of neural stem cell–based therapy in patients with chronic spinal cord injury. Researchers hope that the transplanted stem cells will develop into new neurons that replace severed or lost nerve connections and restore at least some motor and sensory function. 1

Two additional clinical trials at UCSD are testing stem cell–derived therapy for type-1 diabetes and chronic lymphocytic leukemia, the most common form of blood cancer. 1

These three studies are significant in that they are among the first efforts in stem cell research to make the leap from laboratory to human clinical trials. While the number of patients involved in each study is small, researchers are optimistic that as these trials progress and additional trials are launched, a greater number of patients will be enrolled. UCSD reports that trials for heart failure, amyotrophic lateral sclerosis, and blindness are in planning stages. 1

The study of stem cells offers great promise for better understanding basic mechanisms of human development, as well as the hope of harnessing these cells to treat a wide range of diseases and conditions. 2 However, stem cell research— particularly human embryonic stem cell (hESC) research, which involves the destruction of days-old embryos—has also been a source of ongoing ethical, religious, and political controversy. 2

The Politics of Progress

In 1973, the Department of Health, Education, and Welfare (now the Department of Health and Human Services) placed a moratorium on federally funded research using live human embryos. 3 , 4 In 1974, Congress adopted a similar moratorium, explicitly including in the ban embryos created through in vitro fertilization (IVF). In 1992, President George H.W. Bush vetoed legislation to lift the ban, and in 2001, President George W. Bush issued an executive order banning federal funding on stem cells created after that time. 3 , 4 Some states, however, have permitted their limited use. New Jersey, for example, allows the harvesting of stem cells from cloned human embryos, whereas several other states prohibit the creation or destruction of any human embryos for medical research. 3 , 4

In 2009, shortly after taking office, President Barack Obama lifted the eight-year-old ban on federally funded stem cell research, allowing scientists to begin using existing stem cell lines produced from embryos left over after IVF procedures. 5 (A stem cell line is a group of identical stem cells that can be grown and multiplied indefinitely.)

The National Institutes of Health (NIH) Human Embryonic Stem Cell Registry 6 lists the hESCs eligible for use in NIH-funded research. At this writing, 283 eligible lines met the NIH’s strict ethical guidelines for human stem cell research pertaining to the embryo donation process. 7 For instance, to get a human embryonic stem cell line approved, grant applicants must show that the embryos were “donated by individuals who sought reproductive treatment and who gave voluntary written consent for the human embryos to be used for research purposes.” 8 The ESCs used in research are not derived from eggs fertilized in a woman’s body. 9

Because of the separate legislative ban, it is still not possible for researchers to create new hESC lines from viable embryos using federal funds. Federal money may, however, be used to research lines that were derived using private or state sources of funding. 5

While funding restrictions and political debates may have slowed the course of stem cell research in the United States, 10 the field continues to evolve. This is evidenced by the large number of studies published each year in scientific journals on a wide range of potential uses across a variety of therapeutic areas. 11 – 13

The Food and Drug Administration (FDA) has approved numerous stem cell–based treatments for clinical trials. A 2013 report from the Pharmaceutical Research and Manufacturers of America lists 69 cell therapies as having clinical trials under review with the FDA, including 15 in phase 3 trials. The therapeutic categories represented in these trials include cardiovascular disease, skin diseases, cancer and related conditions, digestive disorders, transplantation, genetic disorders, musculoskeletal disorders, and eye conditions, among others. 14

Still, the earliest stem cell therapies are likely years away. To date, the only stem cell–based treatment approved by the FDA for use in this country is for bone marrow transplantation. 15 As of 2010 (the latest year for which data are available), more than 17,000 blood cancer patients had had successful stem cell transplants. 16

A Brief Stem Cell Timeline

Research on stem cells began in the late 19th century in Europe. German biologist Ernst Haeckel coined the term stem cell to describe the fertilized egg that becomes an organism. 17

In the U.S., the study of adult stem cells took off in the 1950s when Leroy C. Stevens, a cancer researcher based in Bar Harbor, Maine, found large tumors in the scrotums of mice that contained mixtures of differentiated and undifferentiated cells, including hair, bone, intestinal, and blood tissue. Stevens and his team concluded that the cells were pluripotent, meaning they could differentiate into any cell found in a fully grown animal. Stem cell scientists are using that carefully documented research today. 17

In 1968, Robert A. Good, MD, PhD, at the University of Minnesota, performed the first successful bone marrow transplant on a child suffering from an immune deficiency. Scientists subsequently discovered how to derive ESCs from mouse embryos and in 1998 developed a method to take stem cells from a human embryo and grow them in a laboratory. 17

Why Stem Cells?

Many degenerative and currently untreatable diseases in humans arise from the loss or malfunction of specific cell types in the body. 9 While donated organs and tissues are often used to replace damaged or dysfunctional ones, the supply of donors does not meet the clinical demand. 18 Stem cells seemingly provide a renewable source of replacement cells and tissues for transplantation and the potential to treat a myriad of conditions.

Stem cells have two important and unique characteristics: First, they are unspecialized and capable of renewing themselves through cell division. When a stem cell divides, each new cell has the potential either to remain a stem cell or to differentiate into other kinds of cells that form the body’s tissues and organs. Stem cells can theoretically divide without limit to replenish other cells that have been damaged. 9

Second, under certain controlled conditions, stem cells can be induced to become tissue- or organ-specific cells with special functions. They can then be used to treat diseases affecting those specific organs and tissues. While bone marrow and gut stem cells divide continuously throughout life, stem cells in the pancreas and heart divide only under appropriate conditions. 9

Embryonic Versus Adult Stem Cells

There are two main types of stem cells: 1) embryonic stem cells (ESCs), found in the embryo at very early stages of development; and 2) somatic or adult stem cells (ASCs), found in specific tissues throughout the body after development. 9

The advantage of embryonic stem cells is that they are pluripotent—they can develop into any of the more than 200 cell types found in the body, providing the potential for a broad range of therapeutic applications. Adult stem cells, on the other hand, are thought to be limited to differentiating into different cell types of their tissue of origin. 9 Blood cells, for instance, which come from adult stem cells in the bone marrow, can specialize into red blood cells, but they will not become other cells, such as neurons or liver cells.

A significant advantage of adult stem cells is that they offer the potential for autologous stem cell donation. In autologous transplants, recipients receive their own stem cells, reducing the risk of immune rejection and complications. Additionally, ASCs are relatively free of the ethical issues associated with embryonic stem cells and have become widely used in research.

Induced Pluripotent Stem Cells

Representing a relatively new area of research, induced pluripotent stem cells (iPSCs) are adult stem cells that have been genetically reprogrammed back to an embryonic stem cell–like state. The reprogrammed cells function similarly to ESCs, with the ability to differentiate into any cell of the body and to create an unlimited source of cells. So iPSCs have significant implications for disease research and drug development.

Pioneered by Japanese researchers in 2006, iPSC technology involves forcing an adult cell, such as a skin, liver, or stomach cell, to express proteins that are essential to the embryonic stem cell identity. The iPSC technology not only bypasses the need for human embryos, avoiding ethical objections, but also allows for the generation of pluripotent cells that are genetically identical to the patient’s. Like adult cells, these unlimited supplies of autologous cells could be used to generate transplants without the risk of immune rejection. 9

In 2013, researchers at the Spanish National Cancer Research Centre in Madrid successfully reprogrammed adult cells in mice, creating stem cells that can grow into any tissue in the body. Prior to this study, iPSCs had never been grown outside Petri dishes in laboratories. 19 And, in July 2013, Japan’s health minister approved the first use of iPSCs in human trials. The Riken Center for Developmental Biology will use the cells to attempt to treat age-related macular degeneration, a common cause of blindness in older people. The small-scale pilot study would test the safety of iPSCs transplanted into patients’ eyes. 20

The Promise of iPSCs

According to David Owens, PhD, Program Director of the Neuroscience Center at NIH’s National Institute of Neurological Disorders and Stroke (NINDS), one of the fundamental hurdles to using stem cells to treat disease is that scientists do not yet fully understand the diseases themselves, that is, the genetic and molecular signals that direct the abnormal cell division and differentiation that cause a particular condition. “You want that before you propose a therapeutic,” he says, “because you want a firm, rational basis for what you’re trying to do, what you’re trying to change.”

Although most of the media attention around stem cells has focused on regenerative medicine and cell therapy, researchers are finding that iPSCs, in particular, hold significant promise as tools for disease modeling. 21 , 22 A major barrier to research is often inaccessibility of diseased tissue for study. 23 Because iPSCs can be derived directly from patients with a given disease, they display all of the molecular characteristics associated with the disease, thereby serving as useful models for the study of pathological mechanisms.

“The biggest payoff early on will be using these cells as a tool to understand the disease better,” says Dr. Owens. For instance, he explains that creating dopamine neurons from iPSC lines could help scientists more closely study the mechanisms behind Parkinson’s disease. “If we get a better handle on the disorders themselves, then that will also help us generate new therapeutic targets.” Recent studies show the use of these patient-specific cells to model other neurodegenerative disorders, including Alzheimer’s and Huntington’s diseases. 24 – 26

In addition to using iPSC technology, it is also possible to derive patient-specific stem cell lines using an approach called somatic cell nuclear transfer (SCNT). This process involves adding the nuclei of adult skin cells to unfertilized donor oocytes. As reported in spring 2014, a team of scientists from the New York Stem Cell Foundation Research Institute and Columbia University Medical Center used SCNT to create the first disease-specific embryonic stem cell line from a patient with type-1 diabetes. The insulin-producing cells have two sets of chromosomes (the normal number in humans) and could potentially be used to develop personalized cell therapies. 27

Many Hurdles Ahead

The development of iPSCs and related technologies may help address the ethical concerns and open up new possibilities for studying and treating disease, but there are still barriers to overcome. One major obstacle is the tendency of iPSCs to form tumors in vivo . Using viruses to genomically alter the cells can trigger the expression of cancer-causing genes, or oncogenes. 28

Much more research is needed to understand the full nature and potential of stem cells as future medical therapies. It is not known, for example, how many kinds of adult stem cells exist or how they evolve and are maintained. 9

Some of the challenges are technical, Dr. Owens explains. For instance, generating large enough numbers of a cell type to provide the amounts needed for treatment is difficult. Some adult stem cells have a very limited ability to divide, making it difficult to multiply them in large numbers. Embryonic stem cells grow more quickly and easily in the laboratory. This is an important distinction because stem cell replacement therapies require large numbers of cells. 29

Also, says Dr. Owens, stem cell transplants present immunological hurdles: “If you do introduce cells into a tissue, will they be rejected if they’re not autologous cells? Or, you might have immunosuppression with the individual who received the cells, and then there are additional complications involved with that. That’s still not entirely clear.”

Such safety issues need to be addressed before any new stem cell–based therapy can advance to clinical trials with real patients. According to Dr. Owens, the preclinical testing stage typically takes about five years. This would include assessment of toxicity, tumorigenicity, and immunogenicity of the cells in treating animal models for disease. 30

“Those are things we have to continually learn about and try to address. It will take time to understand them better,” Dr. Owens says. Asked about the importance of collaboration in overcoming the scientific, regulatory, and financial challenges that lie ahead, he says, “It’s unlikely that one entity could do it all alone. Collaboration is essential.”

Research and Clinical Trials

Ultimately, stem cells have huge therapeutic potential, and numerous studies are in progress at academic institutions and biotechnology companies around the country. Studies at the NIH span multiple disciplines, notes Dr. Owens, who oversees funding for stem cell research at NINDS. ( Figure 1 shows the recent history of NIH funding for stem cell research.) He describes one area of considerable interest as the promotion of regeneration in the brain based on endogenous stem cells. Until recently, it was believed that adult brain cells could not be replaced. However, the discovery of neurogenesis in bird brains in the 1980s led to startling evidence of neural stem cells in the human brain, raising new possibilities for treating neurodegenerative disorders and spinal cord injuries. 31

“It’s a fascinating idea,” says Dr. Owens. “It’s unclear still what the functions of those cells are. They could probably play different roles in different species, but just the fundamental properties themselves are very interesting.” He cites a number of NINDS-funded studies looking at those basic properties.

In another NIH-funded study, Advanced Cell Technology (ACT), a Massachusetts-based biotechnology company, is testing the safety of hESC-derived retinal cells to treat patients with an eye disease called Stargardt’s macular dystrophy. A second ACT trial is testing the safety of hESC-derived retinal cells to treat age-related macular degeneration patients. 32 , 33

In April 2014, scientists at the University of Washington reported that they had successfully regenerated damaged heart muscles in monkeys using heart cells created from hESCs. The research, published in the journal Nature , was the first to show that hESCs can fully integrate into normal heart tissue. 34

The study did not answer every question and had its complications—it failed to show whether the transplanted cells improved the function of the monkeys’ hearts, and some of the monkeys developed arrhythmias. 34 , 35 Still, the researchers are optimistic that it will pave the way for a human trial before the end of the decade and lead to significant advances in treating heart disease. 29

In May 2014, Asterias Biotherapeutics, a California-based biotechnology company focused on regenerative medicine, announced the results of a phase 1 clinical trial assessing the safety of its product AST-OPC1 in patients with spinal cord injuries. 36 The study represents the first-in-human trial of a cell therapy derived from hESCs. Results show that all five subjects have had no serious adverse events associated with the administration of the cells, with the AST-OPC1 itself, or with the immunosuppressive regimen. A phase 1/2a dose-escalation study of AST-OPC1 in patients with spinal cord injuries is awaiting approval from the FDA. 37

The FDA itself has a team of scientists studying the potential of mesenchymal stem cells (MSCs), adult stem cells traditionally found in the bone marrow. Multipotent stem cells, MSCs differentiate to form cartilage, bone, and fat and could be used to repair, replace, restore, or regenerate cells, including those needed for heart and bone repair. 38

Publicly available information about federally and privately funded clinical research studies involving stem cells can be found at http://clinicaltrials.gov . However, the FDA cautions that the information provided on that site is supplied by the product sponsors and is not reviewed or confirmed by the agency.

“The biggest payoff early on will be using these cells as a tool to understand the disease better. If we get a better handle on the disorders themselves, then that will also help us generate new therapeutic targets.” —David Owens, PhD, Program Director, Neuroscience Center, National Institute of Neurological Disorders and Stroke

Global Research Efforts

Stem cell research policy varies significantly throughout the world as countries grapple with the scientific and social implications. In the European Union, for instance, stem cell research using the human embryo is permitted in Belgium, Britain, Denmark, Finland, Greece, the Netherlands, and Sweden; however, it is illegal in Austria, Germany, Ireland, Italy, and Portugal. 39

In those countries where cell lines are accessible, research continues to create an array of scientific advances and widen the scope of stem cell application in human diseases, disorders, and injuries. For example, in February 2014, Cellular Biomedicine Group, a China-based company, released the six-month follow-up data analysis of its phase 1/2a clinical trial for ReJoin, a human adipose-derived mesenchymal precursor cell (haMPC) therapy for knee osteoarthritis. The study, which tested the safety and efficacy of intra-articular injections of autologous haMPCs to reduce inflammation and repair damaged joint cartilage, showed knee pain was significantly reduced and knee mobility was improved. 40 And the journal Stem Cell Research & Therapy reported that researchers at the University of Adelaide in Australia recently completed a project showing stem cells taken from teeth could form “complex networks of brain-like cells.” Although the cells did not grow into full neurons, the researchers say that it will happen given time and the right conditions. 41

The Regulation of Stem Cells

In February 2014, the U.S. Court of Appeals for the District of Columbia Circuit upheld a 2012 ruling that a patient’s stem cells for therapeutic use fall under the aegis of the FDA. 42 The appeals case involved the company Regenerative Sciences, which was using patients’ MSCs in its Regenexx procedure to treat orthopedic problems. 43

The FDA’s Center for Biologics Evaluation and Research (CBER) regulates human cells, tissues, and cellular and tissue-based products (HCT/P) intended for implantation, transplantation, infusion, or transfer into a human recipient, including hematopoietic stem cells. Under the authority of Section 361 of the Public Health Service Act, the FDA has established regulations for all HCT/Ps to prevent the transmission of communicable diseases. 44

The Regenexx case highlights an ongoing debate about whether autologous MSCs are biological drugs subject to FDA approval or simply human cellular and tissue products. Some medical centers collect, concentrate, and reinject MSCs into a patient to treat osteoarthritis but do not add other agents to the injection. The FDA contends that any process that includes culturing, expansion, and added growth factors or antibiotics requires regulation because the process constitutes significant manipulation. Regenerexx has countered that the process does not involve the development of a new drug, which could be given to a number of patients, but rather a patient’s own MSCs, which affects just that one patient.

Ensuring the safety and efficacy of stem cell–based products is a major challenge, says the FDA. Cells manufactured in large quantities outside their natural environment in the human body can potentially become ineffective or dangerous and produce significant adverse effects such as tumors, severe immune reactions, or growth of unwanted tissue. Even stem cells isolated from a person’s own tissue can potentially present these risks when put into an area of the body where they could not perform the same biological function that they were originally performing. Stem cells are immensely complex, the FDA cautions—far more so than many other FDA-regulated products—and they bring with them unique considerations for meeting regulatory standards.

To date, no U.S. companies have received FDA approval for any autologous MSC therapy, although a study is ongoing to assess the feasibility and safety of autologous MSCs for osteoarthritis. 45 One of the major risks with MSCs is that they could potentially lead to cancer or differentiation into bone or cartilage. 46

What’s Next

The numerous stem cell studies in progress across the globe are only a first step on the long road toward eventual therapies for degenerative and life-ending diseases. Because of their unlimited ability to self-renew and to differentiate, embryonic stem cells remain, theoretically, a potential source for regenerative medicine and tissue replacement after injury or disease. However, the difficulty of producing large quantities of stem cells and their tendency to form tumors when transplanted are just a few of the formidable hurdles that researchers still face. In the meantime, the shorter-term payoff of using these cells as a tool to better understand diseases has significant implications.

Social and ethical issues around the use of embryonic stem cells must also be addressed. Many nations, including the U.S., have government-imposed restrictions on either embryonic stem cell research or the production of new embryonic stem cell lines. Induced pluripotent stem cells offer new opportunities for development of cell-based therapies while also providing a way around the ethical dilemma of using embryos, but just how good an alternative they are to embryonic cells remains to be seen.

It is clear that many challenges must be overcome before stem cells can be safely, effectively, and routinely used in the clinical setting. However, their potential benefits are numerous and hold tremendous promise for an array of new therapies and treatments.

Acknowledgments

The authors wish to thank the FDA staff for their support in writing this article and Rachael Conklin, Consumer Safety Officer, Consumer Affairs Branch, Division of Communication and Consumer Affairs, Center for Biologics Evaluation and Research, for her help in organizing the comments provided by FDA staff.

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

Susan Barber Lindquist

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

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

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

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

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

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

Watch: Dr. Mohamad Bydon discusses improvements in research study

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

Stem cells' mechanism of action not fully understood

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

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

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

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

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

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

Research continues into stem cells for spinal cord injuries

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

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

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

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

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

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

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

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• Susan Barber Lindquist, Mayo Clinic Communications, [email protected]
• Personal journey shapes unique perspective Mayo Clinic scientists pioneer immunotherapy technique for autoimmune diseases

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Stem cells: past, present, and future

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Stem Cell Research & Therapy volume  10 , Article number:  68 ( 2019 ) Cite this article

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In recent years, stem cell therapy has become a very promising and advanced scientific research topic. The development of treatment methods has evoked great expectations. This paper is a review focused on the discovery of different stem cells and the potential therapies based on these cells. The genesis of stem cells is followed by laboratory steps of controlled stem cell culturing and derivation. Quality control and teratoma formation assays are important procedures in assessing the properties of the stem cells tested. Derivation methods and the utilization of culturing media are crucial to set proper environmental conditions for controlled differentiation. Among many types of stem tissue applications, the use of graphene scaffolds and the potential of extracellular vesicle-based therapies require attention due to their versatility. The review is summarized by challenges that stem cell therapy must overcome to be accepted worldwide. A wide variety of possibilities makes this cutting edge therapy a turning point in modern medicine, providing hope for untreatable diseases.

Stem cell classification

Stem cells are unspecialized cells of the human body. They are able to differentiate into any cell of an organism and have the ability of self-renewal. Stem cells exist both in embryos and adult cells. There are several steps of specialization. Developmental potency is reduced with each step, which means that a unipotent stem cell is not able to differentiate into as many types of cells as a pluripotent one. This chapter will focus on stem cell classification to make it easier for the reader to comprehend the following chapters.

Totipotent stem cells are able to divide and differentiate into cells of the whole organism. Totipotency has the highest differentiation potential and allows cells to form both embryo and extra-embryonic structures. One example of a totipotent cell is a zygote, which is formed after a sperm fertilizes an egg. These cells can later develop either into any of the three germ layers or form a placenta. After approximately 4 days, the blastocyst’s inner cell mass becomes pluripotent. This structure is the source of pluripotent cells.

Pluripotent stem cells (PSCs) form cells of all germ layers but not extraembryonic structures, such as the placenta. Embryonic stem cells (ESCs) are an example. ESCs are derived from the inner cell mass of preimplantation embryos. Another example is induced pluripotent stem cells (iPSCs) derived from the epiblast layer of implanted embryos. Their pluripotency is a continuum, starting from completely pluripotent cells such as ESCs and iPSCs and ending on representatives with less potency—multi-, oligo- or unipotent cells. One of the methods to assess their activity and spectrum is the teratoma formation assay. iPSCs are artificially generated from somatic cells, and they function similarly to PSCs. Their culturing and utilization are very promising for present and future regenerative medicine.

Multipotent stem cells have a narrower spectrum of differentiation than PSCs, but they can specialize in discrete cells of specific cell lineages. One example is a haematopoietic stem cell, which can develop into several types of blood cells. After differentiation, a haematopoietic stem cell becomes an oligopotent cell. Its differentiation abilities are then restricted to cells of its lineage. However, some multipotent cells are capable of conversion into unrelated cell types, which suggests naming them pluripotent cells.

Oligopotent stem cells can differentiate into several cell types. A myeloid stem cell is an example that can divide into white blood cells but not red blood cells.

Unipotent stem cells are characterized by the narrowest differentiation capabilities and a special property of dividing repeatedly. Their latter feature makes them a promising candidate for therapeutic use in regenerative medicine. These cells are only able to form one cell type, e.g. dermatocytes.

Stem cell biology

A blastocyst is formed after the fusion of sperm and ovum fertilization. Its inner wall is lined with short-lived stem cells, namely, embryonic stem cells. Blastocysts are composed of two distinct cell types: the inner cell mass (ICM), which develops into epiblasts and induces the development of a foetus, and the trophectoderm (TE). Blastocysts are responsible for the regulation of the ICM microenvironment. The TE continues to develop and forms the extraembryonic support structures needed for the successful origin of the embryo, such as the placenta. As the TE begins to form a specialized support structure, the ICM cells remain undifferentiated, fully pluripotent and proliferative [ 1 ]. The pluripotency of stem cells allows them to form any cell of the organism. Human embryonic stem cells (hESCs) are derived from the ICM. During the process of embryogenesis, cells form aggregations called germ layers: endoderm, mesoderm and ectoderm (Fig.  1 ), each eventually giving rise to differentiated cells and tissues of the foetus and, later on, the adult organism [ 2 ]. After hESCs differentiate into one of the germ layers, they become multipotent stem cells, whose potency is limited to only the cells of the germ layer. This process is short in human development. After that, pluripotent stem cells occur all over the organism as undifferentiated cells, and their key abilities are proliferation by the formation of the next generation of stem cells and differentiation into specialized cells under certain physiological conditions.

Oocyte development and formation of stem cells: the blastocoel, which is formed from oocytes, consists of embryonic stem cells that later differentiate into mesodermal, ectodermal, or endodermal cells. Blastocoel develops into the gastrula

Signals that influence the stem cell specialization process can be divided into external, such as physical contact between cells or chemical secretion by surrounding tissue, and internal, which are signals controlled by genes in DNA.

Stem cells also act as internal repair systems of the body. The replenishment and formation of new cells are unlimited as long as an organism is alive. Stem cell activity depends on the organ in which they are in; for example, in bone marrow, their division is constant, although in organs such as the pancreas, division only occurs under special physiological conditions.

Stem cell functional division

Whole-body development.

During division, the presence of different stem cells depends on organism development. Somatic stem cell ESCs can be distinguished. Although the derivation of ESCs without separation from the TE is possible, such a combination has growth limits. Because proliferating actions are limited, co-culture of these is usually avoided.

ESCs are derived from the inner cell mass of the blastocyst, which is a stage of pre-implantation embryo ca. 4 days after fertilization. After that, these cells are placed in a culture dish filled with culture medium. Passage is an inefficient but popular process of sub-culturing cells to other dishes. These cells can be described as pluripotent because they are able to eventually differentiate into every cell type in the organism. Since the beginning of their studies, there have been ethical restrictions connected to the medical use of ESCs in therapies. Most embryonic stem cells are developed from eggs that have been fertilized in an in vitro clinic, not from eggs fertilized in vivo.

Somatic or adult stem cells are undifferentiated and found among differentiated cells in the whole body after development. The function of these cells is to enable the healing, growth, and replacement of cells that are lost each day. These cells have a restricted range of differentiation options. Among many types, there are the following:

Mesenchymal stem cells are present in many tissues. In bone marrow, these cells differentiate mainly into the bone, cartilage, and fat cells. As stem cells, they are an exception because they act pluripotently and can specialize in the cells of any germ layer.

Neural cells give rise to nerve cells and their supporting cells—oligodendrocytes and astrocytes.

Haematopoietic stem cells form all kinds of blood cells: red, white, and platelets.

Skin stem cells form, for example, keratinocytes, which form a protective layer of skin.

The proliferation time of somatic stem cells is longer than that of ESCs. It is possible to reprogram adult stem cells back to their pluripotent state. This can be performed by transferring the adult nucleus into the cytoplasm of an oocyte or by fusion with the pluripotent cell. The same technique was used during cloning of the famous Dolly sheep.

hESCs are involved in whole-body development. They can differentiate into pluripotent, totipotent, multipotent, and unipotent cells (Fig.  2 ) [ 2 ].

Changes in the potency of stem cells in human body development. Potency ranges from pluripotent cells of the blastocyst to unipotent cells of a specific tissue in a human body such as the skin, CNS, or bone marrow. Reversed pluripotency can be achieved by the formation of induced pluripotent stem cells using either octamer-binding transcription factor (Oct4), sex-determining region Y (Sox2), Kruppel-like factor 4 (Klf4), or the Myc gene

Pluripotent cells can be named totipotent if they can additionally form extraembryonic tissues of the embryo. Multipotent cells are restricted in differentiating to each cell type of given tissue. When tissue contains only one lineage of cells, stem cells that form them are called either called oligo- or unipotent.

iPSC quality control and recognition by morphological differences

The comparability of stem cell lines from different individuals is needed for iPSC lines to be used in therapeutics [ 3 ]. Among critical quality procedures, the following can be distinguished:

Short tandem repeat analysis—This is the comparison of specific loci on the DNA of the samples. It is used in measuring an exact number of repeating units. One unit consists of 2 to 13 nucleotides repeating many times on the DNA strand. A polymerase chain reaction is used to check the lengths of short tandem repeats. The genotyping procedure of source tissue, cells, and iPSC seed and master cell banks is recommended.

Identity analysis—The unintentional switching of lines, resulting in other stem cell line contamination, requires rigorous assay for cell line identification.

Residual vector testing—An appearance of reprogramming vectors integrated into the host genome is hazardous, and testing their presence is a mandatory procedure. It is a commonly used procedure for generating high-quality iPSC lines. An acceptable threshold in high-quality research-grade iPSC line collections is ≤ 1 plasmid copies per 100 cells. During the procedure, 2 different regions, common to all plasmids, should be used as specific targets, such as EBNA and CAG sequences [ 3 ]. To accurately represent the test reactions, a standard curve needs to be prepared in a carrier of gDNA from a well-characterized hPSC line. For calculations of plasmid copies per cell, it is crucial to incorporate internal reference gDNA sequences to allow the quantification of, for example, ribonuclease P (RNaseP) or human telomerase reverse transcriptase (hTERT).

Karyotype—A long-term culture of hESCs can accumulate culture-driven mutations [ 4 ]. Because of that, it is crucial to pay additional attention to genomic integrity. Karyotype tests can be performed by resuscitating representative aliquots and culturing them for 48–72 h before harvesting cells for karyotypic analysis. If abnormalities are found within the first 20 karyotypes, the analysis must be repeated on a fresh sample. When this situation is repeated, the line is evaluated as abnormal. Repeated abnormalities must be recorded. Although karyology is a crucial procedure in stem cell quality control, the single nucleotide polymorphism (SNP) array, discussed later, has approximately 50 times higher resolution.

Viral testing—When assessing the quality of stem cells, all tests for harmful human adventitious agents must be performed (e.g. hepatitis C or human immunodeficiency virus). This procedure must be performed in the case of non-xeno-free culture agents.

Bacteriology—Bacterial or fungal sterility tests can be divided into culture- or broth-based tests. All the procedures must be recommended by pharmacopoeia for the jurisdiction in which the work is performed.

Single nucleotide polymorphism arrays—This procedure is a type of DNA microarray that detects population polymorphisms by enabling the detection of subchromosomal changes and the copy-neutral loss of heterozygosity, as well as an indication of cellular transformation. The SNP assay consists of three components. The first is labelling fragmented nucleic acid sequences with fluorescent dyes. The second is an array that contains immobilized allele-specific oligonucleotide (ASO) probes. The last component detects, records, and eventually interprets the signal.

Flow cytometry—This is a technique that utilizes light to count and profile cells in a heterogeneous fluid mixture. It allows researchers to accurately and rapidly collect data from heterogeneous fluid mixtures with live cells. Cells are passed through a narrow channel one by one. During light illumination, sensors detect light emitted or refracted from the cells. The last step is data analysis, compilation and integration into a comprehensive picture of the sample.

Phenotypic pluripotency assays—Recognizing undifferentiated cells is crucial in successful stem cell therapy. Among other characteristics, stem cells appear to have a distinct morphology with a high nucleus to cytoplasm ratio and a prominent nucleolus. Cells appear to be flat with defined borders, in contrast to differentiating colonies, which appear as loosely located cells with rough borders [ 5 ]. It is important that images of ideal and poor quality colonies for each cell line are kept in laboratories, so whenever there is doubt about the quality of culture, it can always be checked according to the representative image. Embryoid body formation or directed differentiation of monolayer cultures to produce cell types representative of all three embryonic germ layers must be performed. It is important to note that colonies cultured under different conditions may have different morphologies [ 6 ].

Histone modification and DNA methylation—Quality control can be achieved by using epigenetic analysis tools such as histone modification or DNA methylation. When stem cells differentiate, the methylation process silences pluripotency genes, which reduces differentiation potential, although other genes may undergo demethylation to become expressed [ 7 ]. It is important to emphasize that stem cell identity, together with its morphological characteristics, is also related to its epigenetic profile [ 8 , 9 ]. According to Brindley [ 10 ], there is a relationship between epigenetic changes, pluripotency, and cell expansion conditions, which emphasizes that unmethylated regions appear to be serum-dependent.

hESC derivation and media

hESCs can be derived using a variety of methods, from classic culturing to laser-assisted methodologies or microsurgery [ 11 ]. hESC differentiation must be specified to avoid teratoma formation (see Fig.  3 ).

Spontaneous differentiation of hESCs causes the formation of a heterogeneous cell population. There is a different result, however, when commitment signals (in forms of soluble factors and culture conditions) are applied and enable the selection of progenitor cells

hESCs spontaneously differentiate into embryonic bodies (EBs) [ 12 ]. EBs can be studied instead of embryos or animals to predict their effects on early human development. There are many different methods for acquiring EBs, such as bioreactor culture [ 13 ], hanging drop culture [ 12 ], or microwell technology [ 14 , 15 ]. These methods allow specific precursors to form in vitro [ 16 ].

The essential part of these culturing procedures is a separation of inner cell mass to culture future hESCs (Fig.  4 ) [ 17 ]. Rosowski et al. [ 18 ] emphasizes that particular attention must be taken in controlling spontaneous differentiation. When the colony reaches the appropriate size, cells must be separated. The occurrence of pluripotent cells lasts for 1–2 days. Because the classical utilization of hESCs caused ethical concerns about gastrulas used during procedures, Chung et al. [ 19 ] found out that it is also possible to obtain hESCs from four cell embryos, leaving a higher probability of embryo survival. Additionally, Zhang et al. [ 20 ] used only in vitro fertilization growth-arrested cells.

Culturing of pluripotent stem cells in vitro. Three days after fertilization, totipotent cells are formed. Blastocysts with ICM are formed on the sixth day after fertilization. Pluripotent stem cells from ICM can then be successfully transmitted on a dish

Cell passaging is used to form smaller clusters of cells on a new culture surface [ 21 ]. There are four important passaging procedures.

Enzymatic dissociation is a cutting action of enzymes on proteins and adhesion domains that bind the colony. It is a gentler method than the manual passage. It is crucial to not leave hESCs alone after passaging. Solitary cells are more sensitive and can easily undergo cell death; collagenase type IV is an example [ 22 , 23 ].

Manual passage , on the other hand, focuses on using cell scratchers. The selection of certain cells is not necessary. This should be done in the early stages of cell line derivation [ 24 ].

Trypsin utilization allows a healthy, automated hESC passage. Good Manufacturing Practice (GMP)-grade recombinant trypsin is widely available in this procedure [ 24 ]. However, there is a risk of decreasing the pluripotency and viability of stem cells [ 25 ]. Trypsin utilization can be halted with an inhibitor of the protein rho-associated protein kinase (ROCK) [ 26 ].

Ethylenediaminetetraacetic acid ( EDTA ) indirectly suppresses cell-to-cell connections by chelating divalent cations. Their suppression promotes cell dissociation [ 27 ].

Stem cells require a mixture of growth factors and nutrients to differentiate and develop. The medium should be changed each day.

Traditional culture methods used for hESCs are mouse embryonic fibroblasts (MEFs) as a feeder layer and bovine serum [ 28 ] as a medium. Martin et al. [ 29 ] demonstrated that hESCs cultured in the presence of animal products express the non-human sialic acid, N -glycolylneuraminic acid (NeuGc). Feeder layers prevent uncontrolled proliferation with factors such as leukaemia inhibitory factor (LIF) [ 30 ].

First feeder layer-free culture can be supplemented with serum replacement, combined with laminin [ 31 ]. This causes stable karyotypes of stem cells and pluripotency lasting for over a year.

Initial culturing media can be serum (e.g. foetal calf serum FCS), artificial replacement such as synthetic serum substitute (SSS), knockout serum replacement (KOSR), or StemPro [ 32 ]. The simplest culture medium contains only eight essential elements: DMEM/F12 medium, selenium, NaHCO 3, l -ascorbic acid, transferrin, insulin, TGFβ1, and FGF2 [ 33 ]. It is not yet fully known whether culture systems developed for hESCs can be allowed without adaptation in iPSC cultures.

Turning point in stem cell therapy

The turning point in stem cell therapy appeared in 2006, when scientists Shinya Yamanaka, together with Kazutoshi Takahashi, discovered that it is possible to reprogram multipotent adult stem cells to the pluripotent state. This process avoided endangering the foetus’ life in the process. Retrovirus-mediated transduction of mouse fibroblasts with four transcription factors (Oct-3/4, Sox2, KLF4, and c-Myc) [ 34 ] that are mainly expressed in embryonic stem cells could induce the fibroblasts to become pluripotent (Fig.  5 ) [ 35 ]. This new form of stem cells was named iPSCs. One year later, the experiment also succeeded with human cells [ 36 ]. After this success, the method opened a new field in stem cell research with a generation of iPSC lines that can be customized and biocompatible with the patient. Recently, studies have focused on reducing carcinogenesis and improving the conduction system.

Retroviral-mediated transduction induces pluripotency in isolated patient somatic cells. Target cells lose their role as somatic cells and, once again, become pluripotent and can differentiate into any cell type of human body

The turning point was influenced by former discoveries that happened in 1962 and 1987.

The former discovery was about scientist John Gurdon successfully cloning frogs by transferring a nucleus from a frog’s somatic cells into an oocyte. This caused a complete reversion of somatic cell development [ 37 ]. The results of his experiment became an immense discovery since it was previously believed that cell differentiation is a one-way street only, but his experiment suggested the opposite and demonstrated that it is even possible for a somatic cell to again acquire pluripotency [ 38 ].

The latter was a discovery made by Davis R.L. that focused on fibroblast DNA subtraction. Three genes were found that originally appeared in myoblasts. The enforced expression of only one of the genes, named myogenic differentiation 1 (Myod1), caused the conversion of fibroblasts into myoblasts, showing that reprogramming cells is possible, and it can even be used to transform cells from one lineage to another [ 39 ].

Although pluripotency can occur naturally only in embryonic stem cells, it is possible to induce terminally differentiated cells to become pluripotent again. The process of direct reprogramming converts differentiated somatic cells into iPSC lines that can form all cell types of an organism. Reprogramming focuses on the expression of oncogenes such as Myc and Klf4 (Kruppel-like factor 4). This process is enhanced by a downregulation of genes promoting genome stability, such as p53. Additionally, cell reprogramming involves histone alteration. All these processes can cause potential mutagenic risk and later lead to an increased number of mutations. Quinlan et al. [ 40 ] checked fully pluripotent mouse iPSCs using whole genome DNA sequencing and structural variation (SV) detection algorithms. Based on those studies, it was confirmed that although there were single mutations in the non-genetic region, there were non-retrotransposon insertions. This led to the conclusion that current reprogramming methods can produce fully pluripotent iPSCs without severe genomic alterations.

During the course of development from pluripotent hESCs to differentiated somatic cells, crucial changes appear in the epigenetic structure of these cells. There is a restriction or permission of the transcription of genes relevant to each cell type. When somatic cells are being reprogrammed using transcription factors, all the epigenetic architecture has to be reconditioned to achieve iPSCs with pluripotency [ 41 ]. However, cells of each tissue undergo specific somatic genomic methylation. This influences transcription, which can further cause alterations in induced pluripotency [ 42 ].

Source of iPSCs

Because pluripotent cells can propagate indefinitely and differentiate into any kind of cell, they can be an unlimited source, either for replacing lost or diseased tissues. iPSCs bypass the need for embryos in stem cell therapy. Because they are made from the patient’s own cells, they are autologous and no longer generate any risk of immune rejection.

At first, fibroblasts were used as a source of iPSCs. Because a biopsy was needed to achieve these types of cells, the technique underwent further research. Researchers investigated whether more accessible cells could be used in the method. Further, other cells were used in the process: peripheral blood cells, keratinocytes, and renal epithelial cells found in urine. An alternative strategy to stem cell transplantation can be stimulating a patient’s endogenous stem cells to divide or differentiate, occurring naturally when skin wounds are healing. In 2008, pancreatic exocrine cells were shown to be reprogrammed to functional, insulin-producing beta cells [ 43 ].

The best stem cell source appears to be the fibroblasts, which is more tempting in the case of logistics since its stimulation can be fast and better controlled [ 44 ].

• Teratoma formation assay

The self-renewal and differentiation capabilities of iPSCs have gained significant interest and attention in regenerative medicine sciences. To study their abilities, a quality-control assay is needed, of which one of the most important is the teratoma formation assay. Teratomas are benign tumours. Teratomas are capable of rapid growth in vivo and are characteristic because of their ability to develop into tissues of all three germ layers simultaneously. Because of the high pluripotency of teratomas, this formation assay is considered an assessment of iPSC’s abilities [ 45 ].

Teratoma formation rate, for instance, was observed to be elevated in human iPSCs compared to that in hESCs [ 46 ]. This difference may be connected to different differentiation methods and cell origins. Most commonly, the teratoma assay involves an injection of examined iPSCs subcutaneously or under the testis or kidney capsule in mice, which are immune-deficient [ 47 ]. After injection, an immature but recognizable tissue can be observed, such as the kidney tubules, bone, cartilage, or neuroepithelium [ 30 ]. The injection site may have an impact on the efficiency of teratoma formation [ 48 ].

There are three groups of markers used in this assay to differentiate the cells of germ layers. For endodermal tissue, there is insulin/C-peptide and alpha-1 antitrypsin [ 49 ]. For the mesoderm, derivatives can be used, e.g. cartilage matrix protein for the bone and alcian blue for the cartilage. As ectodermal markers, class III B botulin or keratin can be used for keratinocytes.

Teratoma formation assays are considered the gold standard for demonstrating the pluripotency of human iPSCs, demonstrating their possibilities under physiological conditions. Due to their actual tissue formation, they could be used for the characterization of many cell lineages [ 50 ].

Directed differentiation

To be useful in therapy, stem cells must be converted into desired cell types as necessary or else the whole regenerative medicine process will be pointless. Differentiation of ESCs is crucial because undifferentiated ESCs can cause teratoma formation in vivo. Understanding and using signalling pathways for differentiation is an important method in successful regenerative medicine. In directed differentiation, it is likely to mimic signals that are received by cells when they undergo successive stages of development [ 51 ]. The extracellular microenvironment plays a significant role in controlling cell behaviour. By manipulating the culture conditions, it is possible to restrict specific differentiation pathways and generate cultures that are enriched in certain precursors in vitro. However, achieving a similar effect in vivo is challenging. It is crucial to develop culture conditions that will allow the promotion of homogenous and enhanced differentiation of ESCs into functional and desired tissues.

Regarding the self-renewal of embryonic stem cells, Hwang et al. [ 52 ] noted that the ideal culture method for hESC-based cell and tissue therapy would be a defined culture free of either the feeder layer or animal components. This is because cell and tissue therapy requires the maintenance of large quantities of undifferentiated hESCs, which does not make feeder cells suitable for such tasks.

Most directed differentiation protocols are formed to mimic the development of an inner cell mass during gastrulation. During this process, pluripotent stem cells differentiate into ectodermal, mesodermal, or endodermal progenitors. Mall molecules or growth factors induce the conversion of stem cells into appropriate progenitor cells, which will later give rise to the desired cell type. There is a variety of signal intensities and molecular families that may affect the establishment of germ layers in vivo, such as fibroblast growth factors (FGFs) [ 53 ]; the Wnt family [ 54 ] or superfamily of transforming growth factors—β(TGFβ); and bone morphogenic proteins (BMP) [ 55 ]. Each candidate factor must be tested on various concentrations and additionally applied to various durations because the precise concentrations and times during which developing cells in embryos are influenced during differentiation are unknown. For instance, molecular antagonists of endogenous BMP and Wnt signalling can be used for ESC formation of ectoderm [ 56 ]. However, transient Wnt and lower concentrations of the TGFβ family trigger mesodermal differentiation [ 57 ]. Regarding endoderm formation, a higher activin A concentration may be required [ 58 , 59 ].

There are numerous protocols about the methods of forming progenitors of cells of each of germ layers, such as cardiomyocytes [ 60 ], hepatocytes [ 61 ], renal cells [ 62 ], lung cells [ 63 , 64 ], motor neurons [ 65 ], intestinal cells [ 66 ], or chondrocytes [ 67 ].

Directed differentiation of either iPSCs or ESCs into, e.g. hepatocytes, could influence and develop the study of the molecular mechanisms in human liver development. In addition, it could also provide the possibility to form exogenous hepatocytes for drug toxicity testing [ 68 ].

Levels of concentration and duration of action with a specific signalling molecule can cause a variety of factors. Unfortunately, for now, a high cost of recombinant factors is likely to limit their use on a larger scale in medicine. The more promising technique focuses on the use of small molecules. These can be used for either activating or deactivating specific signalling pathways. They enhance reprogramming efficiency by creating cells that are compatible with the desired type of tissue. It is a cheaper and non-immunogenic method.

One of the successful examples of small-molecule cell therapies is antagonists and agonists of the Hedgehog pathway. They show to be very useful in motor neuron regeneration [ 69 ]. Endogenous small molecules with their function in embryonic development can also be used in in vitro methods to induce the differentiation of cells; for example, retinoic acid, which is responsible for patterning the nervous system in vivo [ 70 ], surprisingly induced retinal cell formation when the laboratory procedure involved hESCs [ 71 ].

The efficacy of differentiation factors depends on functional maturity, efficiency, and, finally, introducing produced cells to their in vivo equivalent. Topography, shear stress, and substrate rigidity are factors influencing the phenotype of future cells [ 72 ].

The control of biophysical and biochemical signals, the biophysical environment, and a proper guide of hESC differentiation are important factors in appropriately cultured stem cells.

Stem cell utilization and their manufacturing standards and culture systems

The European Medicines Agency and the Food and Drug Administration have set Good Manufacturing Practice (GMP) guidelines for safe and appropriate stem cell transplantation. In the past, protocols used for stem cell transplantation required animal-derived products [ 73 ].

The risk of introducing animal antigens or pathogens caused a restriction in their use. Due to such limitations, the technique required an obvious update [ 74 ]. Now, it is essential to use xeno-free equivalents when establishing cell lines that are derived from fresh embryos and cultured from human feeder cell lines [ 75 ]. In this method, it is crucial to replace any non-human materials with xeno-free equivalents [ 76 ].

NutriStem with LN-511, TeSR2 with human recombinant laminin (LN-511), and RegES with human foreskin fibroblasts (HFFs) are commonly used xeno-free culture systems [ 33 ]. There are many organizations and international initiatives, such as the National Stem Cell Bank, that provide stem cell lines for treatment or medical research [ 77 ].

Stem cell use in medicine

Stem cells have great potential to become one of the most important aspects of medicine. In addition to the fact that they play a large role in developing restorative medicine, their study reveals much information about the complex events that happen during human development.

The difference between a stem cell and a differentiated cell is reflected in the cells’ DNA. In the former cell, DNA is arranged loosely with working genes. When signals enter the cell and the differentiation process begins, genes that are no longer needed are shut down, but genes required for the specialized function will remain active. This process can be reversed, and it is known that such pluripotency can be achieved by interaction in gene sequences. Takahashi and Yamanaka [ 78 ] and Loh et al. [ 79 ] discovered that octamer-binding transcription factor 3 and 4 (Oct3/4), sex determining region Y (SRY)-box 2 and Nanog genes function as core transcription factors in maintaining pluripotency. Among them, Oct3/4 and Sox2 are essential for the generation of iPSCs.

Many serious medical conditions, such as birth defects or cancer, are caused by improper differentiation or cell division. Currently, several stem cell therapies are possible, among which are treatments for spinal cord injury, heart failure [ 80 ], retinal and macular degeneration [ 81 ], tendon ruptures, and diabetes type 1 [ 82 ]. Stem cell research can further help in better understanding stem cell physiology. This may result in finding new ways of treating currently incurable diseases.

Haematopoietic stem cell transplantation

Haematopoietic stem cells are important because they are by far the most thoroughly characterized tissue-specific stem cell; after all, they have been experimentally studied for more than 50 years. These stem cells appear to provide an accurate paradigm model system to study tissue-specific stem cells, and they have potential in regenerative medicine.

Multipotent haematopoietic stem cell (HSC) transplantation is currently the most popular stem cell therapy. Target cells are usually derived from the bone marrow, peripheral blood, or umbilical cord blood [ 83 ]. The procedure can be autologous (when the patient’s own cells are used), allogenic (when the stem cell comes from a donor), or syngeneic (from an identical twin). HSCs are responsible for the generation of all functional haematopoietic lineages in blood, including erythrocytes, leukocytes, and platelets. HSC transplantation solves problems that are caused by inappropriate functioning of the haematopoietic system, which includes diseases such as leukaemia and anaemia. However, when conventional sources of HSC are taken into consideration, there are some important limitations. First, there is a limited number of transplantable cells, and an efficient way of gathering them has not yet been found. There is also a problem with finding a fitting antigen-matched donor for transplantation, and viral contamination or any immunoreactions also cause a reduction in efficiency in conventional HSC transplantations. Haematopoietic transplantation should be reserved for patients with life-threatening diseases because it has a multifactorial character and can be a dangerous procedure. iPSC use is crucial in this procedure. The use of a patient’s own unspecialized somatic cells as stem cells provides the greatest immunological compatibility and significantly increases the success of the procedure.

Stem cells as a target for pharmacological testing

Stem cells can be used in new drug tests. Each experiment on living tissue can be performed safely on specific differentiated cells from pluripotent cells. If any undesirable effect appears, drug formulas can be changed until they reach a sufficient level of effectiveness. The drug can enter the pharmacological market without harming any live testers. However, to test the drugs properly, the conditions must be equal when comparing the effects of two drugs. To achieve this goal, researchers need to gain full control of the differentiation process to generate pure populations of differentiated cells.

Stem cells as an alternative for arthroplasty

One of the biggest fears of professional sportsmen is getting an injury, which most often signifies the end of their professional career. This applies especially to tendon injuries, which, due to current treatment options focusing either on conservative or surgical treatment, often do not provide acceptable outcomes. Problems with the tendons start with their regeneration capabilities. Instead of functionally regenerating after an injury, tendons merely heal by forming scar tissues that lack the functionality of healthy tissues. Factors that may cause this failed healing response include hypervascularization, deposition of calcific materials, pain, or swelling [ 84 ].

Additionally, in addition to problems with tendons, there is a high probability of acquiring a pathological condition of joints called osteoarthritis (OA) [ 85 ]. OA is common due to the avascular nature of articular cartilage and its low regenerative capabilities [ 86 ]. Although arthroplasty is currently a common procedure in treating OA, it is not ideal for younger patients because they can outlive the implant and will require several surgical procedures in the future. These are situations where stem cell therapy can help by stopping the onset of OA [ 87 ]. However, these procedures are not well developed, and the long-term maintenance of hyaline cartilage requires further research.

Osteonecrosis of the femoral hip (ONFH) is a refractory disease associated with the collapse of the femoral head and risk of hip arthroplasty in younger populations [ 88 ]. Although total hip arthroplasty (THA) is clinically successful, it is not ideal for young patients, mostly due to the limited lifetime of the prosthesis. An increasing number of clinical studies have evaluated the therapeutic effect of stem cells on ONFH. Most of the authors demonstrated positive outcomes, with reduced pain, improved function, or avoidance of THA [ 89 , 90 , 91 ].

Rejuvenation by cell programming

Ageing is a reversible epigenetic process. The first cell rejuvenation study was published in 2011 [ 92 ]. Cells from aged individuals have different transcriptional signatures, high levels of oxidative stress, dysfunctional mitochondria, and shorter telomeres than in young cells [ 93 ]. There is a hypothesis that when human or mouse adult somatic cells are reprogrammed to iPSCs, their epigenetic age is virtually reset to zero [ 94 ]. This was based on an epigenetic model, which explains that at the time of fertilization, all marks of parenteral ageing are erased from the zygote’s genome and its ageing clock is reset to zero [ 95 ].

In their study, Ocampo et al. [ 96 ] used Oct4, Sox2, Klf4, and C-myc genes (OSKM genes) and affected pancreas and skeletal muscle cells, which have poor regenerative capacity. Their procedure revealed that these genes can also be used for effective regenerative treatment [ 97 ]. The main challenge of their method was the need to employ an approach that does not use transgenic animals and does not require an indefinitely long application. The first clinical approach would be preventive, focused on stopping or slowing the ageing rate. Later, progressive rejuvenation of old individuals can be attempted. In the future, this method may raise some ethical issues, such as overpopulation, leading to lower availability of food and energy.

For now, it is important to learn how to implement cell reprogramming technology in non-transgenic elder animals and humans to erase marks of ageing without removing the epigenetic marks of cell identity.

Cell-based therapies

Stem cells can be induced to become a specific cell type that is required to repair damaged or destroyed tissues (Fig.  6 ). Currently, when the need for transplantable tissues and organs outweighs the possible supply, stem cells appear to be a perfect solution for the problem. The most common conditions that benefit from such therapy are macular degenerations [ 98 ], strokes [ 99 ], osteoarthritis [ 89 , 90 ], neurodegenerative diseases, and diabetes [ 100 ]. Due to this technique, it can become possible to generate healthy heart muscle cells and later transplant them to patients with heart disease.

Stem cell experiments on animals. These experiments are one of the many procedures that proved stem cells to be a crucial factor in future regenerative medicine

In the case of type 1 diabetes, insulin-producing cells in the pancreas are destroyed due to an autoimmunological reaction. As an alternative to transplantation therapy, it can be possible to induce stem cells to differentiate into insulin-producing cells [ 101 ].

Stem cells and tissue banks

iPS cells with their theoretically unlimited propagation and differentiation abilities are attractive for the present and future sciences. They can be stored in a tissue bank to be an essential source of human tissue used for medical examination. The problem with conventional differentiated tissue cells held in the laboratory is that their propagation features diminish after time. This does not occur in iPSCs.

The umbilical cord is known to be rich in mesenchymal stem cells. Due to its cryopreservation immediately after birth, its stem cells can be successfully stored and used in therapies to prevent the future life-threatening diseases of a given patient.

Stem cells of human exfoliated deciduous teeth (SHED) found in exfoliated deciduous teeth has the ability to develop into more types of body tissues than other stem cells [ 102 ] (Table  1 ). Techniques of their collection, isolation, and storage are simple and non-invasive. Among the advantages of banking, SHED cells are:

Guaranteed donor-match autologous transplant that causes no immune reaction and rejection of cells [ 103 ]

Simple and painless for both child and parent

Less than one third of the cost of cord blood storage

Not subject to the same ethical concerns as embryonic stem cells [ 104 ]

In contrast to cord blood stem cells, SHED cells are able to regenerate into solid tissues such as connective, neural, dental, or bone tissue [ 105 , 106 ]

SHED can be useful for close relatives of the donor

Fertility diseases

In 2011, two researchers, Katsuhiko Hayashi et al. [ 107 ], showed in an experiment on mice that it is possible to form sperm from iPSCs. They succeeded in delivering healthy and fertile pups in infertile mice. The experiment was also successful for female mice, where iPSCs formed fully functional eggs .

Young adults at risk of losing their spermatogonial stem cells (SSC), mostly cancer patients, are the main target group that can benefit from testicular tissue cryopreservation and autotransplantation. Effective freezing methods for adult and pre-pubertal testicular tissue are available [ 108 ].

Qiuwan et al. [ 109 ] provided important evidence that human amniotic epithelial cell (hAEC) transplantation could effectively improve ovarian function by inhibiting cell apoptosis and reducing inflammation in injured ovarian tissue of mice, and it could be a promising strategy for the management of premature ovarian failure or insufficiency in female cancer survivors.

For now, reaching successful infertility treatments in humans appears to be only a matter of time, but there are several challenges to overcome. First, the process needs to have high efficiency; second, the chances of forming tumours instead of eggs or sperm must be maximally reduced. The last barrier is how to mature human sperm and eggs in the lab without transplanting them to in vivo conditions, which could cause either a tumour risk or an invasive procedure.

Therapy for incurable neurodegenerative diseases

Thanks to stem cell therapy, it is possible not only to delay the progression of incurable neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease (AD), and Huntington disease, but also, most importantly, to remove the source of the problem. In neuroscience, the discovery of neural stem cells (NSCs) has nullified the previous idea that adult CNS were not capable of neurogenesis [ 110 , 111 ]. Neural stem cells are capable of improving cognitive function in preclinical rodent models of AD [ 112 , 113 , 114 ]. Awe et al. [ 115 ] clinically derived relevant human iPSCs from skin punch biopsies to develop a neural stem cell-based approach for treating AD. Neuronal degeneration in Parkinson’s disease (PD) is focal, and dopaminergic neurons can be efficiently generated from hESCs. PD is an ideal disease for iPSC-based cell therapy [ 116 ]. However, this therapy is still in an experimental phase ( https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4539501 /). Brain tissue from aborted foetuses was used on patients with Parkinson’s disease [ 117 ]. Although the results were not uniform, they showed that therapies with pure stem cells are an important and achievable therapy.

Stem cell use in dentistry

Teeth represent a very challenging material for regenerative medicine. They are difficult to recreate because of their function in aspects such as articulation, mastication, or aesthetics due to their complicated structure. Currently, there is a chance for stem cells to become more widely used than synthetic materials. Teeth have a large advantage of being the most natural and non-invasive source of stem cells.

For now, without the use of stem cells, the most common periodontological treatments are either growth factors, grafts, or surgery. For example, there are stem cells in periodontal ligament [ 118 , 119 ], which are capable of differentiating into osteoblasts or cementoblasts, and their functions were also assessed in neural cells [ 120 ]. Tissue engineering is a successful method for treating periodontal diseases. Stem cells of the root apical areas are able to recreate periodontal ligament. One of the possible methods of tissue engineering in periodontology is gene therapy performed using adenoviruses-containing growth factors [ 121 ].

As a result of animal studies, dentin regeneration is an effective process that results in the formation of dentin bridges [ 122 ].

Enamel is more difficult to regenerate than dentin. After the differentiation of ameloblastoma cells into the enamel, the former is destroyed, and reparation is impossible. Medical studies have succeeded in differentiating bone marrow stem cells into ameloblastoma [ 123 ].

Healthy dental tissue has a high amount of regular stem cells, although this number is reduced when tissue is either traumatized or inflamed [ 124 ]. There are several dental stem cell groups that can be isolated (Fig.  7 ).

Localization of stem cells in dental tissues. Dental pulp stem cells (DPSCs) and human deciduous teeth stem cells (SHED) are located in the dental pulp. Periodontal ligaments stem cells are located in the periodontal ligament. Apical papilla consists of stem cells from the apical papilla (SCAP)

Dental pulp stem cell (DPSC)

These were the first dental stem cells isolated from the human dental pulp, which were [ 125 ] located inside dental pulp (Table  2 ). They have osteogenic and chondrogenic potential. Mesenchymal stem cells (MSCs) of the dental pulp, when isolated, appear highly clonogenic; they can be isolated from adult tissue (e.g. bone marrow, adipose tissue) and foetal (e.g. umbilical cord) [ 126 ] tissue, and they are able to differentiate densely [ 127 ]. MSCs differentiate into odontoblast-like cells and osteoblasts to form dentin and bone. Their best source locations are the third molars [ 125 ]. DPSCs are the most useful dental source of tissue engineering due to their easy surgical accessibility, cryopreservation possibility, increased production of dentin tissues compared to non-dental stem cells, and their anti-inflammatory abilities. These cells have the potential to be a source for maxillofacial and orthopaedic reconstructions or reconstructions even beyond the oral cavity. DPSCs are able to generate all structures of the developed tooth [ 128 ]. In particular, beneficial results in the use of DPSCs may be achieved when combined with other new therapies, such as periodontal tissue photobiomodulation (laser stimulation), which is an efficient technique in the stimulation of proliferation and differentiation into distinct cell types [ 129 ]. DPSCs can be induced to form neural cells to help treat neurological deficits.

Stem cells of human exfoliated deciduous teeth (SHED) have a faster rate of proliferation than DPSCs and differentiate into an even greater number of cells, e.g. other mesenchymal and non-mesenchymal stem cell derivatives, such as neural cells [ 130 ]. These cells possess one major disadvantage: they form a non-complete dentin/pulp-like complex in vivo. SHED do not undergo the same ethical concerns as embryonic stem cells. Both DPSCs and SHED are able to form bone-like tissues in vivo [ 131 ] and can be used for periodontal, dentin, or pulp regeneration. DPSCs and SHED can be used in treating, for example, neural deficits [ 132 ]. DPSCs alone were tested and successfully applied for alveolar bone and mandible reconstruction [ 133 ].

Periodontal ligament stem cells (PDLSCs)

These cells are used in periodontal ligament or cementum tissue regeneration. They can differentiate into mesenchymal cell lineages to produce collagen-forming cells, adipocytes, cementum tissue, Sharpey’s fibres, and osteoblast-like cells in vitro. PDLSCs exist both on the root and alveolar bone surfaces; however, on the latter, these cells have better differentiation abilities than on the former [ 134 ]. PDLSCs have become the first treatment for periodontal regeneration therapy because of their safety and efficiency [ 135 , 136 ].

Stem cells from apical papilla (SCAP)

These cells are mesenchymal structures located within immature roots. They are isolated from human immature permanent apical papilla. SCAP are the source of odontoblasts and cause apexogenesis. These stem cells can be induced in vitro to form odontoblast-like cells, neuron-like cells, or adipocytes. SCAP have a higher capacity of proliferation than DPSCs, which makes them a better choice for tissue regeneration [ 137 , 138 ].

Dental follicle stem cells (DFCs)

These cells are loose connective tissues surrounding the developing tooth germ. DFCs contain cells that can differentiate into cementoblasts, osteoblasts, and periodontal ligament cells [ 139 , 140 ]. Additionally, these cells proliferate after even more than 30 passages [ 141 ]. DFCs are most commonly extracted from the sac of a third molar. When DFCs are combined with a treated dentin matrix, they can form a root-like tissue with a pulp-dentin complex and eventually form tooth roots [ 141 ]. When DFC sheets are induced by Hertwig’s epithelial root sheath cells, they can produce periodontal tissue; thus, DFCs represent a very promising material for tooth regeneration [ 142 ].

Pulp regeneration in endodontics

Dental pulp stem cells can differentiate into odontoblasts. There are few methods that enable the regeneration of the pulp.

The first is an ex vivo method. Proper stem cells are grown on a scaffold before they are implanted into the root channel [ 143 ].

The second is an in vivo method. This method focuses on injecting stem cells into disinfected root channels after the opening of the in vivo apex. Additionally, the use of a scaffold is necessary to prevent the movement of cells towards other tissues. For now, only pulp-like structures have been created successfully.

Methods of placing stem cells into the root channel constitute are either soft scaffolding [ 144 ] or the application of stem cells in apexogenesis or apexification. Immature teeth are the best source [ 145 ]. Nerve and blood vessel network regeneration are extremely vital to keep pulp tissue healthy.

The potential of dental stem cells is mainly regarding the regeneration of damaged dentin and pulp or the repair of any perforations; in the future, it appears to be even possible to generate the whole tooth. Such an immense success would lead to the gradual replacement of implant treatments. Mandibulary and maxillary defects can be one of the most complicated dental problems for stem cells to address.

Acquiring non-dental tissue cells by dental stem cell differentiation

In 2013, it was reported that it is possible to grow teeth from stem cells obtained extra-orally, e.g. from urine [ 146 ]. Pluripotent stem cells derived from human urine were induced and generated tooth-like structures. The physical properties of the structures were similar to natural ones except for hardness [ 127 ]. Nonetheless, it appears to be a very promising technique because it is non-invasive and relatively low-cost, and somatic cells can be used instead of embryonic cells. More importantly, stem cells derived from urine did not form any tumours, and the use of autologous cells reduces the chances of rejection [ 147 ].

Use of graphene in stem cell therapy

Over recent years, graphene and its derivatives have been increasingly used as scaffold materials to mediate stem cell growth and differentiation [ 148 ]. Both graphene and graphene oxide (GO) represent high in-plane stiffness [ 149 ]. Because graphene has carbon and aromatic network, it works either covalently or non-covalently with biomolecules; in addition to its superior mechanical properties, graphene offers versatile chemistry. Graphene exhibits biocompatibility with cells and their proper adhesion. It also tested positively for enhancing the proliferation or differentiation of stem cells [ 148 ]. After positive experiments, graphene revealed great potential as a scaffold and guide for specific lineages of stem cell differentiation [ 150 ]. Graphene has been successfully used in the transplantation of hMSCs and their guided differentiation to specific cells. The acceleration skills of graphene differentiation and division were also investigated. It was discovered that graphene can serve as a platform with increased adhesion for both growth factors and differentiation chemicals. It was also discovered that π-π binding was responsible for increased adhesion and played a crucial role in inducing hMSC differentiation [ 150 ].

Therapeutic potential of extracellular vesicle-based therapies

Extracellular vesicles (EVs) can be released by virtually every cell of an organism, including stem cells [ 151 ], and are involved in intercellular communication through the delivery of their mRNAs, lipids, and proteins. As Oh et al. [ 152 ] prove, stem cells, together with their paracrine factors—exosomes—can become potential therapeutics in the treatment of, e.g. skin ageing. Exosomes are small membrane vesicles secreted by most cells (30–120 nm in diameter) [ 153 ]. When endosomes fuse with the plasma membrane, they become exosomes that have messenger RNAs (mRNAs) and microRNAs (miRNAs), some classes of non-coding RNAs (IncRNAs) and several proteins that originate from the host cell [ 154 ]. IncRNAs can bind to specific loci and create epigenetic regulators, which leads to the formation of epigenetic modifications in recipient cells. Because of this feature, exosomes are believed to be implicated in cell-to-cell communication and the progression of diseases such as cancer [ 155 ]. Recently, many studies have also shown the therapeutic use of exosomes derived from stem cells, e.g. skin damage and renal or lung injuries [ 156 ].

In skin ageing, the most important factor is exposure to UV light, called “photoageing” [ 157 ], which causes extrinsic skin damage, characterized by dryness, roughness, irregular pigmentation, lesions, and skin cancers. In intrinsic skin ageing, on the other hand, the loss of elasticity is a characteristic feature. The skin dermis consists of fibroblasts, which are responsible for the synthesis of crucial skin elements, such as procollagen or elastic fibres. These elements form either basic framework extracellular matrix constituents of the skin dermis or play a major role in tissue elasticity. Fibroblast efficiency and abundance decrease with ageing [ 158 ]. Stem cells can promote the proliferation of dermal fibroblasts by secreting cytokines such as platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), and basic fibroblast growth factor. Huh et al. [ 159 ] mentioned that a medium of human amniotic fluid-derived stem cells (hAFSC) positively affected skin regeneration after longwave UV-induced (UVA, 315–400 nm) photoageing by increasing the proliferation and migration of dermal fibroblasts. It was discovered that, in addition to the induction of fibroblast physiology, hAFSC transplantation also improved diseases in cases of renal pathology, various cancers, or stroke [ 160 , 161 ].

Oh [ 162 ] also presented another option for the treatment of skin wounds, either caused by physical damage or due to diabetic ulcers. Induced pluripotent stem cell-conditioned medium (iPSC-CM) without any animal-derived components induced dermal fibroblast proliferation and migration.

Natural cutaneous wound healing is divided into three steps: haemostasis/inflammation, proliferation, and remodelling. During the crucial step of proliferation, fibroblasts migrate and increase in number, indicating that it is a critical step in skin repair, and factors such as iPSC-CM that impact it can improve the whole cutaneous wound healing process. Paracrine actions performed by iPSCs are also important for this therapeutic effect [ 163 ]. These actions result in the secretion of cytokines such as TGF-β, interleukin (IL)-6, IL-8, monocyte chemotactic protein-1 (MCP-1), vascular endothelial growth factor (VEGF), platelet-derived growth factor-AA (PDGF-AA), and basic fibroblast growth factor (bFGF). Bae et al. [ 164 ] mentioned that TGF-β induced the migration of keratinocytes. It was also demonstrated that iPSC factors can enhance skin wound healing in vivo and in vitro when Zhou et al. [ 165 ] enhanced wound healing, even after carbon dioxide laser resurfacing in an in vivo study.

Peng et al. [ 166 ] investigated the effects of EVs derived from hESCs on in vitro cultured retinal glial, progenitor Müller cells, which are known to differentiate into retinal neurons. EVs appear heterogeneous in size and can be internalized by cultured Müller cells, and their proteins are involved in the induction and maintenance of stem cell pluripotency. These stem cell-derived vesicles were responsible for the neuronal trans-differentiation of cultured Müller cells exposed to them. However, the research article points out that the procedure was accomplished only on in vitro acquired retina.

Challenges concerning stem cell therapy

Although stem cells appear to be an ideal solution for medicine, there are still many obstacles that need to be overcome in the future. One of the first problems is ethical concern.

The most common pluripotent stem cells are ESCs. Therapies concerning their use at the beginning were, and still are, the source of ethical conflicts. The reason behind it started when, in 1998, scientists discovered the possibility of removing ESCs from human embryos. Stem cell therapy appeared to be very effective in treating many, even previously incurable, diseases. The problem was that when scientists isolated ESCs in the lab, the embryo, which had potential for becoming a human, was destroyed (Fig.  8 ). Because of this, scientists, seeing a large potential in this treatment method, focused their efforts on making it possible to isolate stem cells without endangering their source—the embryo.

Use of inner cell mass pluripotent stem cells and their stimulation to differentiate into desired cell types

For now, while hESCs still remain an ethically debatable source of cells, they are potentially powerful tools to be used for therapeutic applications of tissue regeneration. Because of the complexity of stem cell control systems, there is still much to be learned through observations in vitro. For stem cells to become a popular and widely accessible procedure, tumour risk must be assessed. The second problem is to achieve successful immunological tolerance between stem cells and the patient’s body. For now, one of the best ideas is to use the patient’s own cells and devolve them into their pluripotent stage of development.

New cells need to have the ability to fully replace lost or malfunctioning natural cells. Additionally, there is a concern about the possibility of obtaining stem cells without the risk of morbidity or pain for either the patient or the donor. Uncontrolled proliferation and differentiation of cells after implementation must also be assessed before its use in a wide variety of regenerative procedures on living patients [ 167 ].

One of the arguments that limit the use of iPSCs is their infamous role in tumourigenicity. There is a risk that the expression of oncogenes may increase when cells are being reprogrammed. In 2008, a technique was discovered that allowed scientists to remove oncogenes after a cell achieved pluripotency, although it is not efficient yet and takes a longer amount of time. The process of reprogramming may be enhanced by deletion of the tumour suppressor gene p53, but this gene also acts as a key regulator of cancer, which makes it impossible to remove in order to avoid more mutations in the reprogrammed cell. The low efficiency of the process is another problem, which is progressively becoming reduced with each year. At first, the rate of somatic cell reprogramming in Yamanaka’s study was up to 0.1%. The use of transcription factors creates a risk of genomic insertion and further mutation of the target cell genome. For now, the only ethically acceptable operation is an injection of hESCs into mouse embryos in the case of pluripotency evaluation [ 168 ].

Stem cell obstacles in the future

Pioneering scientific and medical advances always have to be carefully policed in order to make sure they are both ethical and safe. Because stem cell therapy already has a large impact on many aspects of life, it should not be treated differently.

Currently, there are several challenges concerning stem cells. First, the most important one is about fully understanding the mechanism by which stem cells function first in animal models. This step cannot be avoided. For the widespread, global acceptance of the procedure, fear of the unknown is the greatest challenge to overcome.

The efficiency of stem cell-directed differentiation must be improved to make stem cells more reliable and trustworthy for a regular patient. The scale of the procedure is another challenge. Future stem cell therapies may be a significant obstacle. Transplanting new, fully functional organs made by stem cell therapy would require the creation of millions of working and biologically accurate cooperating cells. Bringing such complicated procedures into general, widespread regenerative medicine will require interdisciplinary and international collaboration.

The identification and proper isolation of stem cells from a patient’s tissues is another challenge. Immunological rejection is a major barrier to successful stem cell transplantation. With certain types of stem cells and procedures, the immune system may recognize transplanted cells as foreign bodies, triggering an immune reaction resulting in transplant or cell rejection.

One of the ideas that can make stem cells a “failsafe” is about implementing a self-destruct option if they become dangerous. Further development and versatility of stem cells may cause reduction of treatment costs for people suffering from currently incurable diseases. When facing certain organ failure, instead of undergoing extraordinarily expensive drug treatment, the patient would be able to utilize stem cell therapy. The effect of a successful operation would be immediate, and the patient would avoid chronic pharmacological treatment and its inevitable side effects.

Although these challenges facing stem cell science can be overwhelming, the field is making great advances each day. Stem cell therapy is already available for treating several diseases and conditions. Their impact on future medicine appears to be significant.

After several decades of experiments, stem cell therapy is becoming a magnificent game changer for medicine. With each experiment, the capabilities of stem cells are growing, although there are still many obstacles to overcome. Regardless, the influence of stem cells in regenerative medicine and transplantology is immense. Currently, untreatable neurodegenerative diseases have the possibility of becoming treatable with stem cell therapy. Induced pluripotency enables the use of a patient’s own cells. Tissue banks are becoming increasingly popular, as they gather cells that are the source of regenerative medicine in a struggle against present and future diseases. With stem cell therapy and all its regenerative benefits, we are better able to prolong human life than at any time in history.

Abbreviations

Basic fibroblast growth factor

Bone morphogenic proteins

Dental follicle stem cells

Dental pulp stem cells

Embryonic bodies

Embryonic stem cells

Fibroblast growth factors

Good Manufacturing Practice

Graphene oxide

Human amniotic fluid-derived stem cells

Human embryonic stem cells

Human foreskin fibroblasts

Inner cell mass

Non-coding RNA

Induced pluripotent stem cells

In vitro fertilization

Knockout serum replacement

Leukaemia inhibitory factor

Monocyte chemotactic protein-1

Fibroblasts

Messenger RNA

Mesenchymal stem cells of dental pulp

Myogenic differentiation

Osteoarthritis

Octamer-binding transcription factor 3 and 4

Platelet-derived growth factor

Platelet-derived growth factor-AA

Periodontal ligament stem cells

Rho-associated protein kinase

Stem cells from apical papilla

Stem cells of human exfoliated deciduous teeth

Synthetic Serum Substitute

Trophectoderm

Vascular endothelial growth factor

Transforming growth factors

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Wojciech Zakrzewski, Maria Szymonowicz & Zbigniew Rybak

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Zakrzewski, W., Dobrzyński, M., Szymonowicz, M. et al. Stem cells: past, present, and future. Stem Cell Res Ther 10 , 68 (2019). https://doi.org/10.1186/s13287-019-1165-5

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Study uncovers multiple lineages of stem cells contributing to neuron production

by Elena Garrido, Miguel Hernandez University of Elche

The development of the cerebral cortex largely depends on the stem cells responsible for generating neurons, known as radial glial cells. Until now, it was believed that these stem cells generated neurons following a simple process, that is, a single cell lineage.

However, a study led by the Neurogenesis and cortical expansion laboratory, headed by researcher Víctor Borrell at the Institute for Neurosciences (IN), a joint center of the Spanish National Research Council (CSIC) and the Miguel Hernández University (UMH) of Elche, has discovered not only that there are many more types of radial glial cells than previously thought, but also that there are at least three different processes of neurogenesis that occur in parallel in the same brain areas and at the same moment of development.

The results of this work, published in the journal Science Advances , reveal the complexity of neurogenesis through the involvement of parallel lineages. "We have discovered that there are several alternative routes to generate neurons and that all the routes work at the same time, although we have also seen that the final result is always a neuron with similar characteristics and functions at that stage of development," explains Borrell.

Furthermore, researchers find evidence that the existence of parallel lineages is related to the folding of the cerebral cortex. "A fundamental aspect in this sense is that the 'routes' to form neurons work at the same time and in the same place, but not in the same quantity throughout the cortex, being different between gyrus and sulcus," says the article's first author, Lucía del Valle Antón.

To understand this link, researchers have studied the formation of neurons in regions that will undoubtedly give rise to a gyrus and a sulcus in the ferret brain, while, by using public databases, they have also been able to analyze it in human and mouse brains.

During the development of the study, in which the researcher Juan Antonio Moreno Bravo, who directs the Development, Wiring, and Function of Cerebellar Circuits laboratory, also participated, the experts observed that, although the three lineages are functioning in both gyrus and sulcus zones, different processes predominate depending on the location.

"At first, the cortex is smooth, but there is an area that will grow a lot, and as it grows, it will end up forming a gyrus. Meanwhile, next to it, other areas will grow less and will remain sunken, forming a sulcus," says Borrell. "The first difference between a gyrus and a sulcus is how much it grows, and this is related to how many neurons will be born in that place. For example, in the sulcus, what we find is that of these three 'routes,' the one that generates fewer neurons predominates, while in the gyrus, the opposite will happen."

Understanding the existence of these new types of stem cells , which possess a high capacity for division, along with the various mechanisms for generating neurons in parallel, enables us to comprehend the processes that lead to the enlargement of the human cerebral cortex compared to other species.

This research has allowed scientists to explore, with unprecedented detail, the genes expressed by neurons in both the gyrus and the sulcus. Borrell explains, "We aimed to observe which of all the cells we investigated express genes known to be mutated in human malformations. We verified that not all these cells express the genes responsible for these brain malformations. We observed that they are mainly expressed by the newborn neurons, rather than the progenitors."

Along these lines, the researcher highlights that, despite having the same functions at a global level, the neurons that are born in the gyrus express genes that are essential for the human cortex to have gyri. This indicates that, when patients have malformations because their brain lacks gyri, the defects occur specifically in the neurons of the gyrus and not in those of the sulcus.

International collaboration

In this study, which involved collaboration with researchers from the ISF Stem Cell Research Institute (Helmholtz Zentrum) and the Max Planck Institute for Biological Intelligence, both located in Munich (Germany), the researchers based their results on the sequencing of individual cells at the transcriptomics level, a technique that enables us to identify all the genes that are expressed in each of the cells.

Scientists analyzed thousands of cells using informatics tools to determine the genetic trajectory of these cells and their respective lineages. Upon investigating and validating the lineage data across the three species, they observed that in the human brain, these three parallel lineages also occur, similar to what is observed in ferrets.

However, in the case of mice, analyses conducted have observed only a single predominant route in the creation of neurons. Future research will be necessary to determine whether mice lost these lineages due to evolution or if, on the contrary, these "routes" are still present but in such small proportions that they are undetectable with current tools.

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Paralyzed man who can walk again shows potential benefit of stem cell therapy

A Mayo Clinic study used a patient's stem cells to help repair the spinal cord.

A man who was paralyzed from the neck down after a surfing accident seven years ago is now able to stand and walk on his own , thanks in part to a potentially groundbreaking stem cell treatment.

Chris Barr was the very first patient in a Mayo Clinic study that collected stem cells from his own stomach fat, expanded them in a laboratory to 100 million cells and then injected the cells into Barr's lumbar spine.

Over five years after undergoing the therapy, Barr said he is continuing to gain more independence and get faster at walking.

"I never dreamed I would have a recovery like this," Barr told ABC News' Will Reeve. "I can feed myself. I can walk around. I can do day-to-day independent activities."

Barr shared an update with Reeve on his own progress as Mayo Clinic published new data showing the success of the stem cell treatment in a clinical trial involving 10 patients, including Barr.

According to the trial's results, published Monday in the journal Nature Communications , seven of the 10 patients experienced increased strength in muscle motor groups and increased sensation to pinpricks and light touch.

MORE: Artificial intelligence used in medical procedure to help paralyzed man walk

Three patients in the study had no response to the stem cell therapy, meaning they did not get better or worse, according to the Mayo Clinic, based in Rochester, Minnesota.

"These findings give us hope for the future," Dr. Mohamad Bydon, a neurosurgeon and spinal cord researcher at the Mayo Clinic and the study's lead author, told Reeve, who is also the director of The Christopher Reeve Foundation, a nonprofit "dedicated to curing spinal cord injury," according to its website. The foundation, named in honor of Will Reeve's late father, was not involved in the funding of Bydon's research.

Bydon's research at the Mayo Clinic is a Phase 1 study that began in 2018.

The newly published results of the study show that of the seven patients who saw improvement after the stem cell therapy, each moved up at least one level on the American Spinal Injury Association -- or ASIA -- Impairment Scale, which has five levels documenting patients' function.

"This trial shows us that stem cells are safe and potentially beneficial in the treatment of spinal cord injury," Bydon said. "This can be a milestone in our field of neurosurgery, neuroscience and of treating patients with spinal cord injury."

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Bydon and other researchers are still trying to understand how and why the stem cells interact with the spinal cord to result in progress for some patients, and additional research is underway among a larger group of people to further assess risks and benefits.

In Barr's case, he told Reeve in 2019 he began to quickly see improvements, like getting feeling back in his legs, after undergoing the stem cell treatment.

Now five years later, he described making further long-term improvements, like being able to walk for consistent intervals without assistance.

"I'm just thrilled that there are people taking bold steps to try and do research to cure this," Barr said. "It's been a wild ride and it's not over yet."

Dr. Priscilla Koirala, a member of the ABC News Medical Unit, contributed to this report.

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• Published: 26 April 2023

Regeneration of the heart: from molecular mechanisms to clinical therapeutics

• Qian-Yun Guo 1 ,
• Jia-Qi Yang 1 ,
• Xun-Xun Feng 1 &
• Yu-Jie Zhou 1  

Military Medical Research volume  10 , Article number:  18 ( 2023 ) Cite this article

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Heart injury such as myocardial infarction leads to cardiomyocyte loss, fibrotic tissue deposition, and scar formation. These changes reduce cardiac contractility, resulting in heart failure, which causes a huge public health burden. Military personnel, compared with civilians, is exposed to more stress, a risk factor for heart diseases, making cardiovascular health management and treatment innovation an important topic for military medicine. So far, medical intervention can slow down cardiovascular disease progression, but not yet induce heart regeneration. In the past decades, studies have focused on mechanisms underlying the regenerative capability of the heart and applicable approaches to reverse heart injury. Insights have emerged from studies in animal models and early clinical trials. Clinical interventions show the potential to reduce scar formation and enhance cardiomyocyte proliferation that counteracts the pathogenesis of heart disease. In this review, we discuss the signaling events controlling the regeneration of heart tissue and summarize current therapeutic approaches to promote heart regeneration after injury.

Cardiovascular disease is the leading cause of death and accounts for approximately 32% of global deaths, resulting in the losses of 17.9 million lives each year [ 1 , 2 ]. Military personnel is significantly more likely to report higher work-related stress than civilians [ 3 , 4 ], contributing to the long-term development of cardiovascular diseases and acute triggering of heart failure [ 5 ]. Cardiovascular disease represents the cause of more than 10% of military pilots’ groundings [ 6 ]. The rate of heart failure among hospitalized veterans reaches as high as 0.5% [ 7 ]. These studies highlight the importance of cardiovascular research in military medicine. Despite tremendous efforts and advances in cardiovascular research and therapies, heart failure continues to maintain high mortality and morbidity rates [ 1 , 8 ]. Taking longer life expectancy, higher rates of obesity, diabetes, and modern lifestyle into consideration, epidemiologic studies predicted a 46% increase in heart failure patients by 2030 [ 9 , 10 ]. Figure  1 illustrates the standard of care for managing heart failure. Currently, pharmacological treatment can slow down heart failure progression, but it still needs a breakthrough.

Standard of care for heart failure. Pharmacological treatment and medical devices are currently being used to manage the progression of diseases. β-blocker, ACEi, MRA, and SGLT2i are usually used for all patients with heart failure in order to reduce mortality. For selected patients, diuretic, ivabradine, and digoxin might be used. For advanced stage patients, device and surgery would be recommended. ACEi angiotensin-converting enzyme inhibitor, ARNi angiotensin receptor neprilysin inhibitor, ARB angiotensin receptor blocker, MRA aldosterone receptor antagonists, SGL2i sodium glucose cotransporter 2 inhibitor, HR heart rate, CRT-P cardiac resynchronization therapy-pacemakers, CRT-D cardiac resynchronization therapy-defibrillators, ICD implantable cardioverter defibrillator, MCS mechanical circulatory support

Potential approaches for cardiac regeneration have been tested, including strategies based on in situ cellular reprogramming and de novo tissue engineering methods. Although promising data have been accumulated, each of these approaches faces challenges. Cardiomyocytes of the adult human heart are terminally differentiated and have virtually no regenerative capacity, making it hard to reboot the proliferation of cardiomyocytes after injuries [ 11 ]. Although tissue engineering approaches have developed rapidly owing to the improvement of biomaterials and 3D printing, creating a functional heart in vitro remains a great challenge [ 12 ]. Stem cell-based therapies attempt to promote heart regeneration by injecting stem cells into patients. However, the survival, anchor, differentiation, and maturation of stem cells at the injured site are hard to control, and thus require further optimization before being ready for clinical practice [ 13 , 14 ]. Recent studies suggest that the substances secreted by stem cells may promote heart regeneration [ 15 , 16 ], initiating the search for drugs that target the molecular signaling pathways induced by these substances. Therefore, further understanding the molecular mechanism controlling heart regeneration will help to facilitate the emergence of new therapies that could restore cardiac function. This review summarizes the molecular signaling pathways for heart regeneration and discusses the progress and challenges of approaches for heart regeneration.

Role of molecular signalings in heart regeneration

Notch and notch intracellular domain (nicd) promote cardiomyocyte proliferation and inhibit immune cell infiltration.

Heart regeneration was first described in zebrafish 20 years ago by Poss et al. [ 17 ]. Since this milestone study, the underlying signaling pathways have been extensively studied, as summarized in Fig.  2 , first, showing that Notch mediates heart generation [ 18 ]. Since then, efforts have been made to understand the signaling events boosting cardiomyocyte proliferation, with the hope of aiding human heart regeneration. Notch signaling plays an important role in regulating endocardium maturation via serpine1. Inhibiting or activating Notch both result in impairment of heart regeneration, indicating a dynamic change of Notch activity is crucial [ 19 ]. In addition, Notch signaling in the endocardium interacts with cardiomyocytes as an antagonist for Wnt signaling and promotes cardiomyocyte proliferation [ 20 ]. Following the initial inflammatory response, the endocardium and epicardium regenerate first to provide the right environment for cardiomyocyte proliferation. For example, the endocardium and epicardium secrete retinoic acid, and the epicardium produces fibronectin of extracellular matrix (ECM) [ 21 , 22 ]. The newly-formed heart muscle is found to populate via cardiomyocyte dedifferentiation and proliferation [ 23 ]. A study by Gemberling et al. [ 24 ] demonstrated that neuregulin 1 (Nrg1) is up-regulated after heart injury and serves as a potent inducer of cardiomyocyte proliferation. Notch signaling is also involved in this process, and a remarkable increase in Notch1b and DeltaC expression has been observed [ 18 ]. Interestingly, both Notch inhibition and Notch overexpression have been found to inhibit cardiomyocyte proliferation and heart regeneration, suggesting a delicate balance of this pathway is required [ 25 ]. Further studies by Pfefferli et al. [ 26 ] and Gupta et al. [ 27 ] have distinguished the contribution of different layers of cardiomyocytes during regeneration. Fate mapping with careg:EGFP has shown that the primordial cardiac layer incompletely regenerates after cryoinjury and grow restrictively by lateral expansion, while cortical and trabecular layers are primarily responsible for myocardium growth. When overexpressed specifically in cardiomyocytes, Notch also improves cardiac function by reducing the formation of scars [ 28 ]. Notch signaling pathway as a potential target for therapeutic approaches has been recently discussed [ 29 ]. Functional screening of congenital heart disease risk loci shows that maml3 mutants can decrease cardiomyocyte proliferation through inhibition of Notch signaling [ 30 ], indicating that overexpression of maml3 may induce cardiomyocyte proliferation by activating Notch.

Signaling pathways in heart repair and regeneration. Hippo-YAP, Notch and Nrg-ErbB signaling pathways are the major players in regulating heart repair and regeneration after injuries. Hippo-Yap regulates cardiomyocyte proliferation, migration, and apoptosis, thus affecting scar formation after injury. Notch signaling controls cardiomyocyte proliferation, as well as immune cell infiltration and endocardial cell maturation. Nrg-ErbB signaling affects cardiomyocyte dedifferentiation, division, and survival. FAT4 FAT atypical cadherin 4, MST macrophage stimulating, SAV1 salvador family domain-containing protein 1, LATS large tumor suppressor kinase, MOB1 MOB kinase activator 1, YAP Yes-associated protein, TAZ tafazzin, phospholipid-lysophospholipid transacylase, TEAD TEA domain family, ADAM ADAM metallopeptidase domain, NICD Notch intracellular domain, MAM mastermind, CSL citrate synthase like, ErbB2 Erb-B2 receptor tyrosine kinase, RAF v-raf-leukemia viral oncogene, PI3K phosphatidylinositol 3-kinase, MEK1 mitogen-activated protein kinase kinase 1, ERK extracellular signal-regulated kinase, Akt protein kinase B, mTOR mechanistic target of rapamycin kinase, JUN Jun proto-oncogene, ETS ETS transcription factor family, FOS FBJ osteosarcoma oncogene, LRP LDL receptor related protein, GSK-3β glycogen synthase kinase-3 beta, TCF T-cell factor, LEF lymphoid enhancer factor

Hippo and Yes-associated protein (YAP) regulate cardiomyocyte proliferation and scar formation

The Hippo-YAP pathway is highly conserved and plays a pivotal role in cardiomyocyte cell cycle re-entry. Hippo deficiency enhances cardiomyocyte regeneration and heart functional recovery while reducing scar formation after myocardial infarction in adult mice [ 31 , 32 ]. The Hippo-deficient cardiomyocytes express higher levels of proliferative and stress response genes, such as Park2 [ 32 ]. YAP, the inactivated downstream effector of Hippo, is abundant in neonatal heart tissue but not in adult heart tissue. Recent studies found YAP to be a key regulator for cardiac development and regeneration in mice [ 33 , 34 , 35 ]. Similar to inhibiting Hippo, activation of YAP results in less scar formation and improved heart function after myocardial infarction at postnatal days 7 and 28 as well as adult stages [ 35 , 36 ]. In Erb-B2 receptor tyrosine kinase (ErbB2)-overexpressed mice, YAP mediated a robust epithelial-mesenchymal transition (EMT)-like regeneration by interacting with the cytoskeleton and altering the mechanical characteristics of the cell [ 33 ]. In addition, non-coding RNAs make up a major part of the complex regulatory signaling network. Eulalio et al. [ 37 ] found that miR-199a and miR-590 can effectively induce cell cycle re-entry of cardiomyocytes in vitro as well as in neonatal and adult mice. In murine myocardial infarction models, overexpression of miR-199a and miR-590 via single-dose injection of synthetic RNA promotes cardiac regeneration and recovery of cardiac function [ 37 , 38 ]. Recently, Gabisonia et al. [ 39 ] found that, using infarcted pig hearts, miR-199a was shown to facilitate cardiac repair and increase muscle mass and contractility. Follow-up studies on miR-199a have identified potential downstream signaling, such as CD151 [ 40 ], mechanistic target of rapamycin (mTOR) [ 41 ] and Wnt2 [ 42 ]. Cardiac-specific overexpression of miR-128 in neonatal mice attenuates cardiomyocyte proliferation and functional recovery after myocardial infarction. miR-128 regulates cardiomyocyte cell cycle re-entry via SUZ12, a chromatin modifier that targets p27, cyclin E, and CK2 [ 43 ]. Overexpression of miR-195 (a member of miR-15) leads to reduced proliferation and hypertrophy of cardiomyocytes, while inhibition of the miR-15 family increases cardiomyocyte proliferation after myocardial infarction in adult mice. The downstream target of miR-195 includes cell cycle genes, mitochondrial genes, and inflammatory genes [ 44 ]. Similarly, miR-1/-133a is also a negative regulator of cardiomyocyte cell cycle re-entry in the adult heart. Short-term deletion of miR-1/-133a protects against myocardial infarction. However, long-term deficiency leads to heart failure [ 45 ]. circNfix, a circular RNA, is up-regulated in the adult hearts of humans and mice. Knocking down circNfix releases suppression on downstream cyclinA2 and cyclinB1 and increases miR-214 activity, leading to enhanced cardiomyocyte proliferation and recovery after injury [ 46 ]. miR-152 has been found to be a target of Toll-like receptor 3 (TLR3) and induces cardiomyocyte proliferation by regulating cell cycle proteins downstream of YAP1 [ 47 ]. Recent study shows that FAM122A, an endogenous inhibitor of protein phosphatase 2A, is a novel regulator in the mesendodermal specification and cardiac differentiation via Hippo and Wnt signaling pathways [ 48 ]. In the first step, RNA-binding protein LIN28a stimulates the formation of new cardiomyocytes and prevents cardiomyocyte apoptosis [ 49 ]. Activation of YAP promotes progenitor regeneration by triggering LIN28a transcription [ 50 ].

To date, little is understood about the removal of the scar and the functional integration of regenerated cardiomyocytes. The collagenolytic activity was detected in the injured region between day 14 to 30. In the same period, expressions of matrix metalloproteins (MMPs), such as MMP-2 and MMP-14a, are up-regulated, suggesting a potential role for them in scar removal [ 51 ]. Expression of miR-101a is inhibited after the onset of injury but up-regulated between days 7 to 14. Suppression of miR-101a promotes cardiomyocyte proliferation but inhibits scar removal. Depletion of the downstream target gene Fosab rescued the scar-clearing defect of miR-101a inhibition, demonstrating that miR-101a regulates scar removal via Fosab [ 52 ]. Scar formation is regulated by YAP signaling, and macrophages directly produce collagen to make up the scar [ 53 , 54 ]. Deletion of YAP from zebrafish does not affect the proliferation of cardiomyocytes but leads to larger injuries, showing that initial scar formation is important to control the damage [ 53 ]. In zebrafish, fibrosis does not preclude scar-free regeneration [ 55 , 56 ].

ErbB/PI3K/ERK and Wnt/β-catenin control cardiomyocyte proliferation, dedifferentiation, and inflammation

The Nrg1/ErbB has been recognized as a potential signaling pathway involved in the heart regeneration program. Nrg1 was initially proposed for its potential relevance to mitogenic effects in mammalian cardiomyocytes and further was proved in the post-injured zebrafish heart by Gemberling et al. [ 24 ], which provided the foundation for mouse experiments and clinical trials. In adult mice, injection of Nrg1 induces cell cycle re-entry and cardiomyocyte division. Inactivation of the tyrosine receptor ErbB4 for Nrg1 reduces cardiomyocyte proliferation, while stimulation of ErbB4 enhances it [ 57 ]. The deletion of another co-receptor for Nrg1, ErbB2, also shows its importance for cardiomyocyte proliferation in neonatal mice. Constitutive activation of ErbB2 in both neonatal and adult mice leads to cardiomyocyte proliferation and dedifferentiation via extracellular signal-regulated kinase (ERK), protein kinase B (Akt) and glycogen synthase kinase-3 beta (GSK-3β)/β-catenin downstream signaling. Notably, transient activation of ErbB2 promotes regeneration after myocardial infarction in mice [ 58 ].

The initial inflammatory response is required for complete regenerative capacity. Anti-inflammatory treatment reduces cardiomyocyte proliferation and impairs the vascularization of newly-formed tissue, resulting in an inability to clear the fibrotic deposition [ 59 ]. In contrast, the immune cell is not required for cardiomyocyte mitotic activity under normal conditions [ 59 ]. Fang et al. [ 60 ] have found that inflammatory cytokines promote cardiomyocyte proliferation via activating JAK1/STAT3 signaling. Inhibiting this signaling by expressing a dominant negative form of STAT3 leads to a reduction in cardiomyocyte proliferation. MAPK/ERK acts as a critical signaling for vertebrate tissue regeneration; its potential roles in tissue engineering and regenerative medicine have been emphasized [ 61 ]. Kynurenine stimulates cardiomyocyte proliferation by activating the cytoplasmic aryl hydrocarbon receptor-SRC-YAP/ERK pathway; it also stimulates cardiac angiogenesis by facilitating aryl hydrocarbon receptor nuclear translocation and increasing vascular endothelial growth factor A (VEGF-A) expression [ 62 ]. Dual-specificity phosphatase 6 (DUSP6), which can dephosphorylate ERK1/2, is a regenerative repressor during zebrafish heart regeneration [ 63 ]. Deletion of Dusp6 in mice improves cardiac outcomes by reducing neutrophil-mediated myocardial damage induced by myocardial infarction-caused inflammation [ 64 ]. Furthermore, a DUSP6 inhibitor has been tested in myocardial infarction rats, showing that it improves heart function and suppresses inflammatory cardiac remodeling [ 65 ]. In addition, the cardiac-derived ECM may provide an ideal scaffold for heart tissue engineering [ 66 ], and nuclear pore numbers are decreased during cardiomyocyte maturation, and this reduces nuclear responses to activation of MAPK induced by extracellular signals [ 67 ]. Activation of Nrf1, a stress-responsive transcription factor is seen in regenerating cardiomyocytes. Nrf1 overexpression can protect the heart from ischemic injury, while deletion inhibits neonatal heart regeneration by affecting proteasome and redox balance [ 28 ]. The role of Wnt in promoting cardiomyocyte differentiation has been further investigated, showing that it may provide a powerful tool for stem cell-based regeneration therapy [ 68 ]. These studies suggest that the molecular events initiated by extracellular signals may have therapeutic benefits for heart regeneration.

Approaches and challenges for heart regeneration

The fate mapping experiments in mice have shown that new cardiomyocytes originate from pre-existing ones, during homeostasis [ 69 ], after injury in adults [ 69 , 70 ], and during neonatal heart regeneration [ 71 ]. In addition, using a transgenic line of hypoxia-inducible factor-1α (HIF-1α), Kimura et al. [ 70 ] showed that hypoxic cardiomyocytes exhibit characteristics of neonatal heart cells and contribute mostly to cardiomyocyte formation in adults. Despite these results, many efforts have been focused on the c-Kit + progenitor cells from the bone marrow [ 72 ], which were later shown to play a negligible role in heart regeneration [ 73 ]. Using the Cre/lox system and a reporter line, endogenous c-Kit + cells are found to generate cardiomyocytes at a percentage less than 0.03. Although c-Kit + cells contribute to the revascularization of cardiac endothelial cells, their role in myocardium regeneration is insignificant.

In order to develop new therapies, recent studies have worked on understanding the regulatory role of non-muscle cells, such as immune cells, endothelial cells, and cardiac fibroblasts. In neonatal mice, CD4 + regulatory T cells (Tregs) are necessary for cardiac regeneration. Depletion of Tregs inhibits cardiomyocyte proliferation and induces fibrosis, whereas adoptive transfer of Tregs rescues this phenotype [ 74 ]. Interestingly, ablation of CD4 + Tregs in mice at postnatal day 8 promotes heart regeneration after resection [ 75 ], suggesting the role of immune cells might differ by stages. Endothelial cells support heart regeneration by reassembling arteries, which serve as a scaffold for cardiomyocyte repopulation and also reperfuse the ischemic tissues [ 76 , 77 ]. Endothelial cell migration is induced by the CXCL12-CXCR4 signaling pathway. Genetic inhibition of this signaling leads to formation of larger scars and the reduction of cardiomyocyte proliferation after myocardial infarction [ 76 ]. Consistent with this, inhibition of revascularization in zebrafish with dominant negative VEGF-A also hindered regeneration, suggesting that endothelial cells are actively engaged in cardiomyocyte proliferation [ 78 ]. Cardiac fibroblasts deposit ECM and their number increases during development and diseases, such as heart failure [ 79 ]. Transcriptomic analysis showed different gene expression profiles between fetal and adult fibroblasts of humans, suggesting fibroblasts might be potential contributors to embryonic heart regeneration [ 80 ]. However, ablating activated fibroblasts in mice has a protective effect after acute injuries [ 81 ], which contradicts its vital function in promoting heart regeneration of zebrafish [ 82 ]. This could potentially be explained by the existence of different sub-clusters of fibroblasts in the heart, but further studies are still needed [ 83 ]. In summary, modulating immune cells, endothelial cells, and fibroblasts after injury may promote cardiac regeneration and lead to further mitigation of disease.

The regenerative ability of the mammalian heart is lost during development. In humans, the scar-free repair of the heart is feasible, but only at early developmental stages [ 84 , 85 ]. A case report of a newborn child by Haubner et al. [ 86 ] showed strong regeneration ability after severe myocardial infarction and tissue damage. The cardiac function of this 1-year-old child recovered a few weeks after the initial injury. Similar responses have been seen in other cases by Cesna et al. [ 87 ], Deutsch et al. [ 88 ], and Farooqi et al. [ 89 ], leading to the hope of repairing a damaged adult heart by reactivating regenerative processes that are present during the neonatal stage. Similar to humans, mice lose the capacity for heart regeneration during the early postnatal stage between postnatal days 1 to 7 [ 84 ]. A well-designed study by Drenckhahn et al. [ 90 ] showed that embryonic cardiomyocytes are able to re-enter the cell cycle and proliferate to form heart muscles. In this study, the X-linked gene Hccs was deleted specifically in the heart muscle; this deletion is lethal for the cell. In heterozygous females (half of the cardiomyocytes were normal due to random X inactivation), the mutant cells contributed to less than 10% of tissue volume, showing that the normal cardiomyocytes are able to regenerate about 50% loss of cardiomyocytes at embryonic stage [ 90 ]. By removing 10% of the ventricle from mice at various ages, the time windows of regeneration are characterized [ 71 ]. The murine heart can regenerate at postnatal day 1 after surgical resection with minimal scar or hypertrophy [ 91 ]. This regenerative ability is continuously lost until it ceases at postnatal day 7. In support of this conclusion, similar results have been observed in many other injury models by Haubner et al. [ 92 ] and Porrello et al. [ 44 ] although the collagen scar has been observed when resecting a larger part (20%) of the ventricle [ 93 ]. A study by Porrello et al. [ 44 ] using left anterior descending artery (LAD) ligation-induced injury showed that the heart regenerates within 3 weeks after extensive necrosis. This study compared changes in gene expression after injury between postnatal days 1, 3, and 10. Many genes regulating mitosis, cell division, cell cycle, and ECM synthesis have been identified, including NPPA (atrial natriuretic factor), Nanog (stem cell marker), and HIF3A (hypoxia-inducible factor-3α gene) [ 92 ]. Further study by Darehzereshki et al. [ 91 ] with cryoinjury models has revealed different modes of repair after different types of injury. Neonatal hearts are able to regenerate after non-transmural cryoinjury but not after transmural injury and differential plasminogen activator inhibitor 1 (PAI-1) expression could be a potential explanation. Konfino et al. [ 94 ] found that both neonatal and adult mice respond differently to LAD-induced myocardial infarction and resection. The adult heart forms a thin scar after myocardial infarction, whereas apical resection leads to the occurrence of a hemorrhagic scar. Together, these findings suggest that different treatments should be developed to administer to specific injuries.

The limitation of this model is the lack of cell death, inflammation, and debris clearance steps during the healing process [ 95 ]. Cryoinjury is one of the most commonly used methods, in which a precooled metal is used to freeze part of the ventricle [ 55 , 56 ]. Although cardiac tissue loss is similar to the resection model, it takes much longer, around 130 d, to regenerate the heart after cryoinjury [ 56 ]. Genetic models of cardiomyocyte death have also been used to study heart regeneration in zebrafish. Wang et al. [ 96 ] ablated cardiomyocyte with the expression of cytotoxic diphtheria toxin A chain, induced by cell-specific cyclization recombination enzyme (Cre). This method induces around 60% loss of cardiomyocytes while leaving the endocardium and epicardium intact, which resembles cardiomyopathy in human patients [ 97 ]. Heart function and tissue are restored in around 30 d, which could be attributed to the importance of epicardium in heart regeneration [ 98 ].

Using these injury models, the cellular processes of heart regeneration have been better characterized and a signaling network of genes was identified to be crucial for scar-free regeneration. The regenerative process can be separated into four major stages: 1) the acute reaction to injury, including recruitment of immune cells and deposition of fibrotic tissues; 2) the endocardium and epicardium regenerate in order to support the myocardium; 3) the myocardium is regenerated via proliferation, and 4) the functional integration of newly generated cardiomyocytes, scar removal, and inflammation resolution [ 95 ].

Transplantation of progenitor-derived cells and stem cells

Cell transplantation to repair the injured heart has been started for more than a decade. Intracoronary administration of bone marrow-derived progenitor cells can improve the recovery of left ventricular contractile function in patients with acute myocardial infarction [ 99 ]. However, studies with double-blind randomized designs show that injection of bone marrow mononuclear cells fails to improve the left ventricular contractile function [ 100 , 101 , 102 ]. The randomized placebo-controlled study of myoblast transplantation also shows that myoblast injections are unable to improve echocardiographic heart function [ 103 ]. Adverse effects such as arrhythmias are always problematic, as skeletal myoblasts are not able to conduct electromechanical signals as cardiomyocytes [ 104 ]. Therefore, efficient treatment may be cell-specific and achieved by transplantation of progenitor-derived cells. Recent studies have graded mesoderm assembly controls cell fate and morphogenesis of the early mammalian heart [ 105 ].

Another approach is to induce the differentiation of cardiomyocytes in vitro using embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). Both cell types are able to succeed in vitro to produce cardiomyocyte-like cells [ 106 , 107 ]. Convincing evidence shows that transplantation of ESC-derived cardiomyocytes improves heart function by integrating with pre-existing cardiomyocytes to transduce electromechanical signals [ 108 , 109 ]. Although transplantation of human ESC-derived cardiomyocytes can regenerate the infarcted pig heart, it induces ventricular tachyarrhythmias [ 110 ]. There have been few clinical trials in humans given the ethical challenges of ESCs as well as concerns about side effects. One trial shows some positive results, but with an overall low engraft rate and lack of careful characterization of the control group [ 111 ]. Similarly, another trial shows that transplantation of iPSC-derived cardiomyocytes improves ventricular contractility and promotes heart regeneration, but has low engraftment and survival rate of cardiomyocytes, and induces complications such as tachycardia [ 112 , 113 ].

The POSEIDON study shows that bone marrow-derived mesenchymal stem cells (MSCs) may have cardiogenic potential and improve the functional capacity of the heart [ 114 ]. However, the conclusion is hindered by the lack of a placebo control group and a small patient cohort of 30. However, a randomized double-blind trial shows that bone marrow-derived mesenchymal stromal cells produce a moderate improvement in left ventricular ejection fraction (LVEF) and stroke volume of ischemic heart [ 115 ]. Similar results have been reported in trials using MSCs derived from different sources, such as the umbilical cord-mesenchymal stem cell (UC-MSC) [ 116 ]. The Congestive Heart Failure Cardiopoietic Regenerative Therapy (CHART-1) trial demonstrated that MSC injection is overall safe [ 117 ] and has long-term benefits in patients with significant left ventricular enlargement [ 118 , 119 ]. The recent CONCERT-HF trial shows that MSC in combination with c-Kit positive cells (CPCs) can significantly reduce heart failure-related major adverse cardiac events (HF-MACE). However, no improvement in left ventricular function or reduction of scar size can be achieved, requiring further elucidation of the underlying mechanism [ 120 , 121 ]. Other clinical trials show that MSC injection fails to produce functional improvement of the heart [ 122 , 123 ]. Although MSCs can differentiate into cardiomyocytes in vitro [ 124 ], MSC-derived endothelial cells are the main contributor to heart regeneration in animal model [ 125 ]. A randomized double-blind multi-center trial TEAM-AMI shows that the efficacy of MSC injection is highly dependent on the microenvironment [ 126 ], supporting that the clinical benefits are mainly mediated by indirect effects instead of by generating new cardiomyocytes [ 123 ]. Vagnozzi et al. [ 127 ] showed that intracardiac injection of killed stem cells or use of chemical inducers for immune response produced similar results as live adult stem cell. All these treatments induce a regional accumulation of CCR2 + and CX3CR1 + macrophages, which affect fibroblasts and the ECM at the injury site. A series of animal studies by Bolli et al. [ 128 ] demonstrated that transplanted cells cannot engraft into the myocardium nor differentiate to cardiomyocytes, although improved cardiac function was observed. This dissociation of therapeutic improvement with engrafting rate has been seen among MSCs, ESCs, and CPCs treatment, independent of delivery method and preconditions [ 129 ]. These new findings suggest that the benefits from stem cell injection are mainly due to secreted factors instead of cell replenishment. Therefore, understanding the molecular signaling induced by factors secreted by stem cells becomes more important for treatment of heart injury. Recent studies show that endoderm-derived islet1-expressing cells can differentiate into endothelial cells to function as hematopoietic stem and progenitor cells [ 130 ], which may serve as an alternative approach for stem cell transplant; in addition, human- or animal-derived decellularized heart patches have been used in vivo and in vitro studies to promote the regeneration of heart tissue [ 131 ]. However, due to the complexity of cardiac tissue engineering, significant hard work must be done before the approaches can be clinically used.

Currently, a growing number of clinical trials [ 130 ] (see Bolli et al. [ 129 ] for a comprehensive list of trials) and Meta-analyses [ 132 ] have greatly expanded the knowledge and potential choices of cell sources and interventions for heart disease, such as IMMNC-HF with bone marrow mononuclear cell [ 133 ]; LAPiS (NCT04945018), HEAL-CHF (NCT03763136) and NCT05223894 with human iPSC derived cardiomyocytes; NCT05147766 with UC-MSCs; NCT03797092 with adipose-derived stromal cell; and BioVAT-HF (NCT04396899) with engineered human myocardium. DREAM-HF, a phase III clinical trial, recruited 565 patients and upon completion will provide evidence in analyzing the efficiency of MSC injection as a heart failure treatment [ 14 , 134 , 135 ]. Recent studies show that human mesenchymal stromal cells and endothelial colony-forming cells reduce cardiomyocyte apoptosis, scar size, and adverse cardiac remodeling, compared to vehicle, in a pre-clinical model of acute myocardial infarction [ 136 ]. Human ESC-derived endothelial cells also attenuate cardiac remodeling in a mouse myocardial infarction model [ 137 ]. Besides cardiomyocytes, cardiac interstitial cells also play crucial roles during cardiac regeneration [ 138 ], which opens another avenue to improve heart regeneration. These studies provide useful information for cell therapy approach to treat cardiac injury in the future.

Inducing proliferation of existing cardiomyocytes

The safest and least immunogenic option for cardiac regeneration is using pre-existing cardiomyocytes, although human cardiomyocytes are well-known for being non-proliferative [ 85 ]. There is evidence supporting that cardiomyocytes self-renew at a slow but steady speed [ 69 ], and previous mechanistic studies in mice and zebrafish have provided clues for potential therapeutic targets. Combined expression of cell cycle-related genes, Cdk1 , Ccnb , Cdk4, and Ccnd induces post-mitotic cell proliferation and improves ventricular function after myocardial infarction [ 139 ]. As discussed earlier, the Hippo-YAP pathway is a promising target for promoting cardiomyocyte proliferation. Adeno-associated virus (AAV)-based genetic knockdown of Hippo pathway gene Sav in pig models has been shown to increase the renewal rate of cardiomyocytes after myocardial infarction and improve LVEF [ 140 ]. No arrythmia, tumor formation, or mortality has occurred after treatment, making this a promising approach to advancing clinical trials.

Another potential target is Myc, a transcription factor involved in cell replication, differentiation, metabolism, and apoptosis [ 141 ]. Four-hour acute activation of Myc signaling in juvenile mice leads to a marked proliferative response in vivo [ 142 ]. Mechanistically, this effect is mediated by positive transcription elongation factor b (P-TEFb), which consists of CDK9 and cyclinT1. Furthermore, a transient cardiomyocyte-specific expression of Myc, SRY-box transcription factor 2 (SOX2), OCT4 (named POU5F1; POU domain, class 5, transcription factor 1), and KLF transcription factor 4 (KLF4) can induce dedifferentiation of adult cardiomyocytes characterized by a gene expression profile resembling that of fetal cells. This allows the reprogrammed cardiomyocytes to re-enter the cell cycle and divide into more cardiomyocytes, leading to improved cardiac function after myocardial infarction [ 143 ]. Prolonged expression of these four factors resulted in tumor formation and lethality in mice, however, urging the need for more in-depth studies to avoid potential safety issues.

The Nrg1 has shown its mitogenic effect in pre-existing cardiomyocytes (mentioned in section “ErbB/PI3K/ERK and Wnt/β-catenin control cardiomyocyte proliferation, dedifferentiation, and inflammation”). Furthermore, Polizzotti et al. [ 144 ] show that recombinant neuregulin 1 (rNRG1) induces the proliferation of cardiomyocytes both in mice and in isolated human myocardium, which opened the therapeutic window and prompted clinical trials. A double-blind, placebo-controlled clinical trial of neuregulin 1β3 (cimaglermin alfa) shows sustained improvements in LVEF [ 145 ]. Another clinical trial shows that recombinant human neuregulin 1 (rhNRG1) can increase LVEF and decrease end-diastolic volume (EDV) and end-systolic volume (ESV) in chronic heart failure patients. However, these results were statistically indistinguishable from the placebo, and it remains unclear if this treatment improves heart function by inducing regeneration [ 146 ]. Overall, there is active research underway to develop and optimize therapies using identified gene targets and to explore new targets, i.e. , Hoxb13 by Nguyen et al. [ 147 ], Meis1 by Mahmoud et al. [ 148 ], and miR-199a by Eulalio et al. [ 37 ] and Gabisonia et al. [ 39 ].

Reprogramming non-muscle cells into cardiomyocytes

Reprogramming other cells of the heart, such as fibroblasts, into cardiomyocytes, is another way to achieve the challenging task of repairing the heart. As a large cell population of the heart [ 149 ], fibroblasts are the first responders after cardiac injuries, thus making them an ideal source of cardiomyocytes. Forced expression of cardiac transcription factor combinations, such as GATA binding protein 4 (GATA4), myocyte enhancer factor 2C (MEF2C), and T-box transcription factor 5 (TBX5) (GMT cocktail) [ 150 ]; or GATA4, heart and neural crest derivatives expressed transcript 2 (HAND2), MEF2C and TBX5 (GHMT) [ 151 ], can successfully transform fibroblasts into cardiomyocytes in vitro. Bypassing the iPSC stage, this approach reprograms fibroblasts directly into contractile cardiomyocytes that express typical cardiomyocyte markers. In vivo expression of GHMT using retroviral infection in mice showed that reprogramed cells can form cardiomyocytes and conduct electromechanical signals after myocardial infarction induced by LAD ligation [ 151 ]. Many genes and signaling pathways involved in heart regeneration also modulate reprogramming of fibroblast into cardiomyocytes, including Notch signaling [ 152 ], zinc finger transcription factor 281 (ZNF281; regulating inflammation) [ 153 ], fibroblast growth factor (FGF) and VEGF [ 154 ], Akt1/protein kinase B [ 155 ], Bmi1 (epigenetic factor) [ 156 ], and chemical factors [ 157 ]. Recently, Wang et al. [ 158 ] found that autophagic factor Beclin1 negatively regulates fibroblast reprogramming in an autophagy-independent manner, and that Beclin1 haploinsufficiency in mice promotes reprogramming and reduces scar size after myocardial infarction. In addition, a combination of miRNAs, miR-1, -133, -208, and -499 have also been found to induce cardiomyocytes from fibroblasts both in vitro and in vivo [ 159 , 160 ], providing alternative targets for fibroblast reprogramming. Alternatively, Lalit et al. [ 161 ] showed that mesoderm posterior bHLH transcription factor 1 (MESP1), GATA4, TBX5, NK2 homeobox 5 (NKX2-5), and BAF60C (SMARCD3, SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily D, member 3) expressed in fibroblasts produce a progenitor population that gives rise to cardiomyocytes, endothelial cells, and mural cells in myocardial infarction mice models. Recent study also suggests that the cardiac gene TBX20 (T-box transcription factor 20) enhances myocardial reprogramming induced by the MGT133 reprogramming cocktail (MEF2C, GATA4, TBX5, and miR-133) [ 162 ]. In summary, transcription factor combinations play an important role in transforming fibroblasts into cardiomyocytes in mice.

Despite the success in mice, human fibroblasts are more resistant to both the transcription factor and miRNA combination-induced reprogramming and have shown overall inadequate efficacy to produce cardiomyocytes. Furthermore, the induced cardiomyocytes mostly lack contractility [ 163 , 164 ]. Follow-up studies discovered that the reprogramming process of human fibroblasts requires the addition of other factors, such as MESP1 and myocardin (MYOCD) [ 165 , 166 ], ZFPM2 (zinc finger protein, FOG family member 2) [ 166 ], V-Ets erythroblastosis virus E26 oncogene homolog 2 (ETS2) and MESP1 [ 167 ]. More efforts are still needed to understand the molecular mechanism and the heterogeneity [ 168 ] of induced cardiomyocytes and improve the efficacy of this approach before clinical application. Nevertheless, studies using mouse models have reached a new level by using a novel Tcf21iCre/reporter/MGTH2A transgenic mouse system showing that cardiac reprogramming can repair myocardial infarction [ 169 ]. However, whether it is safe and efficacy for patients remains to be validated.

Non-cell-based approaches

Although still in the early stages, approaches that are not based on cells have the great potential as they bypass the difficulties related to low engraft rates, unclear mechanism, and ethical and safety problems. Study by Puente et al. [ 170 ] in postnatal mice found that oxidative stress induces cell cycle arrest, thus contributing to the loss of heart regenerative ability. Based on this finding, Nakada et al. [ 171 ] designed experiments where mice were exposed to hypoxia for a week after myocardial infarction. This treatment triggers a robust regenerative response and improves left ventricular systolic response. Fate-mapping showed that pre-existing cardiomyocytes proliferate to form myocardium, making it an intriguing idea to treat patients with gradual systemic hypoxia.

Secreted factors, such as growth factors VEGF-A, FGF-2, Nrg1, and thymosin b4, protect against myocardial injuries in animal models [ 172 , 173 ]. However, this effect has not been seen in clinical trials with both VEGF-A and FGF-2 [ 174 , 175 ]. One explanation for this might be that the delivery method cannot ensure a high bioavailability, as a better recovery is achieved by using synthetic mRNA to express VEGF-A in mice [ 176 ]. Recent studies show that VEGF-A-induced angiogenic sprouting can be attenuated by siRNA knockdown or CRISPR/Cas9 knockout of LINC00607 [ 177 ]. VEGF mRNA has been administrated to patients via direct intramyocardial injection, showing that it may be safe for introducing genetic material to the cardiac muscle [ 178 ]. Nrg1 sustains the epicardial-mediated cardiac regeneration capacity of neonatal heart explants [ 179 ]. Oxytocin also activates epicardial cells and promotes heart regeneration after cardiac injury [ 180 ]. Daily administration of thymosin β4, a peptide known to restore vascularization of the epicardium [ 181 ], gives mice the capability of producing new cardiomyocytes and improves recovery after myocardial infarction [ 182 , 183 ]. These studies have been confirmed by a recent report showing that thymosin β4 and also prothymosin α promote cardiac regeneration in mice [ 184 ]. Exosomes are small extracellular vesicles containing different cargoes like protein, RNA and lipids [ 185 ]. Exosomes secreted by iPSC or cardiac progenitor populations promote cardiac functional recovery in animal models [ 186 , 187 ]. Furthermore, mechanistic studies by Cai et al. [ 188 ] and Zhou et al. [ 189 ] showed that the epicardium, similar to stromal stem cell, plays an important role in heart regeneration by both serving as a source for cardiomyocytes, and most importantly, by providing the required paracrine factors [ 190 ]. A proteomic study by Arrell et al. [ 191 ] comparing chronic infarction models with and without human stem cell treatment identified 283 and 450 altered proteins, respectively. This finding could provide a roadmap to future therapeutics using secreted factors. Owing to the advancement of the biomedical engineering field, new methods are being developed to efficiently deliver these factors, including exosomes [ 192 ], cardiac patches [ 193 ], and bioactive hydrogel [ 194 ]. For example, a recent report shows that cardiac tissue regeneration can be induced by the delivery of miR-126 and miR-146a via exosomes [ 195 ]. Recent studies show that cardiogel-loaded chitosan patches or injectable hydrogels containing anti-apoptotic, anti-inflammatory, and pro-angiogenic agents may have therapeutic benefits for heart injury [ 196 , 197 ]. Together, the precise delivery of factors promoting myocardial proliferation and inhibiting apoptosis and inflammation has the potential to enable heart regeneration in situ.

Together, these findings provide exciting new directions for regenerative therapeutics for human heart disease. Notably, there are several barriers that need to be removed before translating these findings to clinical practice, such as the variability between species and the insufficient reproduction of results [ 198 ]. By using quantitative measurement, human-animal chimeras [ 199 ], large-animal models and platforms, i.e., CIBERCV Cardioprotection Large Animal Platform (CIBER-CLAP) [ 198 , 200 ], standardized protocols and quality-control infrastructure [ 201 ], future preclinical studies are anticipated to yield positive clinical results.

Conclusions and perspectives

In summary, active research in the field has revealed common molecular mechanisms for heart regeneration and potential new targets for therapies. These potential gene targets function to regulate immune response, cardiac fibroblast activation, epicardium recovery, and cardiomyocyte proliferation after injuries. Inspired by these findings, current trials focus on inducing heart regenerative ability by cell-based approaches, including progenitor cell transplantation, inducing cardiomyocyte proliferation, and direct reprogramming. Other ongoing therapeutic explorations involve non-cell-based approaches, such as secreted factors and exosomes. In addition, the contribution of non-cardiomyocytes, such as endothelial cells and the epicardium has been actively studied. Figure  3 illustrates current approaches for heart regeneration. With studies for genetics and genomics developed gradually, gene editing technology, especially CRISPR/Cas9, has made continuous breakthroughs, which opens up a new way to manipulate the genome in vitro and in vivo, and also provides an unprecedented opportunity to explore the application of gene editing in cardiovascular diseases [ 202 , 203 ]. iPSCs are increasingly being used as substitutes or supplements for animal models of cardiovascular disease [ 204 ]. Jiang et al. [ 205 ] have found that fibroblasts could be reprogrammed into cardiovascular progenitor cells using transgenic methods, which are called CRISPR-induced cardiovascular progenitor cells (ciCPCs). The implanted ciCPCs differentiate into cardiovascular cells in vivo, which significantly improve myocardial systolic function and the formation of scars, and provide a new source of cells for myocardial regeneration. With the development of artificial intelligence, Theodoris et al. [ 206 ] recently developed a machine learning approach to identify small molecules, which correct gene networks dysregulated in iPSC broadly. This approach could prevent and treat specific cardiovascular diseases in a mouse model. This study points to human–machine learning, network analysis, and iPSC technology to make this strategy feasible and potentially represent an effective path for drug discovery [ 206 ]. In addition, Lin et al. [ 207 ] demonstrated that multiplexed CRISPRi screening combined with machine learning confers functional robustness to gene expression. The prediction of synergistic enhancers by machine learning provides an effective strategy for identifying pairs of noncoding variants associated with disease-causing genes beyond the analysis of genome-wide association studies [ 207 ]. There’s a reasonable prospect that gene editing and artificial intelligence will also bring breakthroughs in heart regeneration in the future. These attempts generated promising results and could be further optimized and then tested in larger populations. Cre recombinase microinjection will help researchers identify the cell progenitors and gene networks involved in organ development [ 208 ]. A variety of tissues and organs including hearts have been produced via 3D bio-printing, which serves as in vitro models for pharmacokinetics and drug screening [ 209 ]. Although it is not promised, 3D bio-printing may be used for repairing, or even replacing, an injured heart in the future. We believe that the endeavors in fighting against heart injury will finally lead to a breakthrough for adult heart regeneration.

Current approaches for heart regeneration. Current attempts at heart regenerative therapies include cell based and non-cell based approaches. Each of these approaches has its own advantages and faces different challenges. iPSC induced pluripotent stem cell, BMC bone marrow cell, MSC mesenchymal stem cell, Cdk1 cyclin-dependent kinase 1, Ccnb cyclin B, SOX2 SRY-box transcription factor 2, OCT4 POU domain, class 5, transcription factor 1, KLF4 KLF transcription factor 4, YAP Yes-associated protein, Nrg neuregulin, FGF fibroblast growth factor, VEGF vascular endothelial growth factor, GATA4 GATA binding protein 4, HAND2 heart and neural crest derivatives expressed transcript 2, MEF2C Myocyte enhancer factor 2C, TBX5 T-box transcription factor 5

Availability of data and materials

Not applicable.

Abbreviations

Adeno-associated virus

Protein kinase B

SMARCD3, SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily d, member 3

Bone marrow cells

Congestive Heart Failure Cardiopoietic Regenerative Therapy

CIBERCV Cardioprotection Large Animal Platform

CRISPR-induced cardiovascular progenitor cells

C-Kit positive cells

Extracellular matrix

End-diastolic volume

Epithelial-mesenchymal transition

Erb-B2 receptor tyrosine kinase

Extracellular signal-regulated kinase

Embryonic stem cells

End-systolic volume

Fibroblast growth factor

GATA binding protein 4

Glycogen synthase kinase-3 beta

Heart and neural crest derivatives expressed transcript 2

Heart failure-related major adverse cardiac events

Hypoxia-inducible factor 3α gene

Hypoxia-inducible factor-1α

Induced pluripotent stem cells

KLF transcription factor 4

Left anterior descending artery

Left ventricular ejection fraction

Myocyte enhancer factor 2C

Mesoderm posterior bHLH transcription factor 1

Mesenchymal stem cell

Mechanistic target of rapamycin kinase

Notch intracellular domain

NK2 homeobox 5

Neuregulin 1

POU domain, class 5, transcription factor 1

Positive transcription elongation factor b

Recombinant human neuregulin 1

Recombinant neuregulin 1

SRY-box transcription factor 2

T-box transcription factor 5

Regulatory T cells

Toll-like receptor 3

Umbilical cord-mesenchymal stem cell

Vascular endothelial growth factor A

Yes-associated protein

Zinc finger protein, FOG family member 2

Zinc finger transcription factor 281

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Acknowledgements

We want to thank Michael Simons from Yale Cardiovascular Center for providing the learning opportunity for Qian-Yun Guo. In addition, we thank the biorender.com for providing platform of creating research graphics.

This work was supported by the Natural Science Foundation of Beijing, China (7214223, 7212027), the Beijing Hospitals Authority Youth Programme (QML20210601), the Chinese Scholarship Council (CSC) scholarship (201706210415), the National Key Research and Development Program of China (2017YFC0908800), the Beijing Municipal Health Commission (PXM2020_026272_000002, PXM2020_026272_000014), and the National Natural Science Foundation of China (82070293).

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Guo, QY., Yang, JQ., Feng, XX. et al. Regeneration of the heart: from molecular mechanisms to clinical therapeutics. Military Med Res 10 , 18 (2023). https://doi.org/10.1186/s40779-023-00452-0

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Study lays the basis for new knowledge on gastrointestinal diseases

The meeting point of the stomach and esophagus, the so-called gastro-esophageal junction, is a region of the human body that is not well-suited to the modern lifestyle. Stress, alcohol, nicotine and severe obesity are often triggers for pathological changes to the mucosal membrane in this area, often resulting in esophageal cancer.

An international research team has now gained new insights into the development of the cells, their communication with each other, and their regulation at the junction of the esophagus and stomach. With the help of specially developed mini-organs, so-called organoids, and with techniques that make it possible to track and profile individual cells, they have been able to follow the development of the gastro-esophageal junction from embryonic to adult stage in detail using animal experiments.

New Insights into the Development of the Gastrointestinal Tract

Their results reveal the complex communication at the cellular level and the specific pathways that these cells use to communicate. They provide new insights into the development of the gastro-esophageal junction and thus have significant implications for the understanding, prevention and treatment of gastrointestinal diseases. At the same time, they present new starting points for medical research and the development of new therapies.

Cindrilla Chumduri is responsible for this study, which has now been published in the journal Nature Communications . Until recently, the infection and cancer biologist was a research group leader at the Department of Microbiology at Julius-Maximilians-Universität Würzburg (JMU); she is now an associate professor at Aarhus University (Denmark). Other participants came from Charité -- Universitätsmedizin and the Max Planck Institute for Infection Biology in Berlin.

"This collaboration underlines the importance of different expertise to improve our understanding of the biology of the gastrointestinal tract," says Chumduri. She herself has many years of experience in research with organoids. Among other things, she has used mini-organs she developed to study how cells in the cervix degenerate and turn cancerous -- another region where different types of mucosal cells collide.

Where Different Epithelia Meet

"The squamous epithelia of the esophagus and the columnar epithelia of the stomach meet at the gastroesophageal junction," explains Dr. Naveen Kumar Nirchal, one of the first authors of the study. The area is known as a "hotspot for the development of metaplasia" -- the replacement of one type of cell by another.

Barrett's esophagus, a precursor to esophageal cancer, often develops there, the number of cases of which has increased dramatically in the Western world over the past four decades. "Barrett's esophagus is characterized by the replacement of the resident squamous epithelium of the esophagus by other cell types that are not normally found in this tissue," says the scientist.

However, it is still unclear why this region is so susceptible to this process. In order to better understand this transformation, it is therefore first necessary to decipher the normal development process in detail -- from embryo to mature adult. "This is the only way to determine the tissue changes that trigger the progression of the disease, explains Dr. Rajendra Kumar Gurumurthy, another researcher of the study.

A Never-Before-Seen Insight into the Development of this Region

This has now been achieved: By using a novel approach that combines organoid and mouse models with advanced single-cell transcriptome analyses over time and space, the research team has shed light on the complex developmental process of the gastroesophageal junction. "We were able to provide unprecedented insight into the development of this region from the embryonic stage to adulthood in mice and identify the intricate composition of the cells involved and how they develop," explains Pon Ganish Prakash, another scientist involved in the study.

The work shows the sophisticated communication between different cell types within the gastroesophageal junction and the signaling pathways involved. "This understanding opens up new avenues for research into gastrointestinal diseases," says Cindrilla Chumduri.

Above all, the precision of the single-cell analysis in their study opens new doors to understanding how pathological processes develop and to developing innovative treatments, the team writes in its study. The work will therefore be a "cornerstone for understanding the development of such diseases" and will significantly influence the approach to the early detection and treatment of diseases in this important part of the digestive system.

• Gastrointestinal Problems
• Diseases and Conditions
• Nervous System
• Lung Cancer
• Immune System
• Stomach cancer
• Athletic training
• Sleep disorder
• Ovarian cancer

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Materials provided by University of Würzburg . Original written by Gunnar Bartsch. Note: Content may be edited for style and length.

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• Naveen Kumar, Pon Ganish Prakash, Christian Wentland, Shilpa Mary Kurian, Gaurav Jethva, Volker Brinkmann, Hans-Joachim Mollenkopf, Tobias Krammer, Christophe Toussaint, Antoine-Emmanuel Saliba, Matthias Biebl, Christian Jürgensen, Bertram Wiedenmann, Thomas F. Meyer, Rajendra Kumar Gurumurthy, Cindrilla Chumduri. Decoding spatiotemporal transcriptional dynamics and epithelial fibroblast crosstalk during gastroesophageal junction development through single cell analysis . Nature Communications , 2024; 15 (1) DOI: 10.1038/s41467-024-47173-z

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