stem cell research

NIH Stem Cell Information

Introduction to stem cells.

• What are stem cells, and why are they important?
• What are the unique properties of all stem cells?
• How do you culture stem cells in the laboratory?
• How are stem cells used in biomedical research and therapies?
• How does NIH support stem cell research?

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Stem cells: what they are and what they do.

Stem cells offer promise for new medical treatments. Learn about stem cell types, current and possible uses, and the state of research and practice.

You've heard about stem cells in the news, and perhaps you've wondered if they might help you or a loved one with a serious disease. Here are some answers to frequently asked questions about stem cells.

What are stem cells?

Stem cells: The body's master cells

Stem cells are the body's master cells. All other cells arise from stem cells, including blood cells, nerve cells and other cells.

Stem cells are a special type of cells that have two important properties. They are able to make more cells like themselves. That is, they self-renew. And they can become other cells that do different things in a process known as differentiation. Stem cells are found in almost all tissues of the body. And they are needed for the maintenance of tissue as well as for repair after injury.

Depending on where the stem cells are, they can develop into different tissues. For example, hematopoietic stem cells reside in the bone marrow and can produce all the cells that function in the blood. Stem cells also can become brain cells, heart muscle cells, bone cells or other cell types.

There are various types of stem cells. Embryonic stem cells are the most versatile since they can develop into all the cells of the developing fetus. The majority of stem cells in the body have fewer abilities to give rise to cells and may only help maintain and repair the tissues and organs in which they reside.

No other cell in the body has the natural ability to generate new cell types.

Why is there such an interest in stem cells?

Researchers are studying stem cells to see if they can help to:

• Increase understanding of how diseases occur. By watching stem cells mature into cells in bones, heart muscle, nerves, and other organs and tissue, researchers may better understand how diseases and conditions develop.

Generate healthy cells to replace cells affected by disease (regenerative medicine). Stem cells can be guided into becoming specific cells that can be used in people to regenerate and repair tissues that have been damaged or affected by disease.

People who might benefit from stem cell therapies include those with leukemia, Hodgkin disease, non-Hodgkin lymphoma and some solid tumor cancers. Stem cell therapies also might benefit people who have aplastic anemia, immunodeficiencies and inherited conditions of metabolism.

Stem cells are being studied to treat type 1 diabetes, Parkinson's disease, amyotrophic lateral sclerosis, heart failure, osteoarthritis and other conditions.

Stem cells may have the potential to be grown to become new tissue for use in transplant and regenerative medicine. Researchers continue to advance the knowledge on stem cells and their applications in transplant and regenerative medicine.

Test new drugs for safety and effectiveness. Before giving drugs in development to people, researchers can use some types of stem cells to test the drugs for safety and quality. This type of testing may help assess drugs in development for toxicity to the heart.

New areas of study include the effectiveness of using human stem cells that have been programmed into tissue-specific cells to test new drugs. For the testing of new drugs to be accurate, the cells must be programmed to acquire properties of the type of cells targeted by the drug. Techniques to program cells into specific cells are under study.

Where do stem cells come from?

There are several sources of stem cells:

Embryonic stem cells. These stem cells come from embryos that are 3 to 5 days old. At this stage, an embryo is called a blastocyst and has about 150 cells.

These are pluripotent (ploo-RIP-uh-tunt) stem cells, meaning they can divide into more stem cells or can become any type of cell in the body. This allows embryonic stem cells to be used to regenerate or repair diseased tissue and organs.

• Adult stem cells. These stem cells are found in small numbers in most adult tissues, such as bone marrow or fat. Compared with embryonic stem cells, adult stem cells have a more limited ability to give rise to various cells of the body.

Adult cells altered to have properties of embryonic stem cells. Scientists have transformed regular adult cells into stem cells using genetic reprogramming. By altering the genes in the adult cells, researchers can make the cells act similarly to embryonic stem cells. These cells are called induced pluripotent stem cells (iPSCs).

This new technique may allow use of reprogrammed cells instead of embryonic stem cells and prevent immune system rejection of the new stem cells. However, scientists don't yet know whether using altered adult cells will cause adverse effects in humans.

Researchers have been able to take regular connective tissue cells and reprogram them to become functional heart cells. In studies, animals with heart failure that were injected with new heart cells had better heart function and survival time.

Perinatal stem cells. Researchers have discovered stem cells in amniotic fluid as well as umbilical cord blood. These stem cells can change into specialized cells.

Amniotic fluid fills the sac that surrounds and protects a developing fetus in the uterus. Researchers have identified stem cells in samples of amniotic fluid drawn from pregnant women for testing or treatment — a procedure called amniocentesis.

Why is there controversy about using embryonic stem cells?

The National Institutes of Health created guidelines for human stem cell research in 2009. The guidelines define embryonic stem cells and how they may be used in research and include recommendations for the donation of embryonic stem cells. Also, the guidelines state that embryonic stem cells from embryos created by in vitro fertilization can be used only when the embryo is no longer needed.

Where do these embryos come from?

The embryos being used in embryonic stem cell research come from eggs that were fertilized at in vitro fertilization clinics but never implanted in women's uteruses. The stem cells are donated with informed consent from donors. The stem cells can live and grow in special solutions in test tubes or petri dishes in laboratories.

Why can't researchers use adult stem cells instead?

Progress in cell reprogramming and the formation of iPSCs has greatly enhanced research in this field. However, reprogramming is an inefficient process. When possible, iPSCs are used instead of embryonic stem cells since this avoids the ethical issues about use of embryonic stem cells that may be morally objectionable for some people.

Although research into adult stem cells is promising, adult stem cells may not be as versatile and durable as are embryonic stem cells. Adult stem cells may not be able to be manipulated to produce all cell types, which limits how adult stem cells can be used to treat diseases.

Adult stem cells are also more likely to contain irregularities due to environmental hazards, such as toxins, or from errors acquired by the cells during replication. However, researchers have found that adult stem cells are more adaptable than was first thought.

What are stem cell lines, and why do researchers want to use them?

A stem cell line is a group of cells that all descend from a single original stem cell and are grown in a lab. Cells in a stem cell line keep growing but don't become specialized cells. Ideally, they remain free of genetic defects and continue to create more stem cells. Clusters of cells can be taken from a stem cell line and frozen for storage or shared with other researchers.

What is stem cell therapy (regenerative medicine), and how does it work?

Stem cell therapy, also known as regenerative medicine, promotes the repair response of diseased, dysfunctional or injured tissue using stem cells or their derivatives. It is the next chapter in organ transplantation and uses cells instead of donor organs, which are limited in supply.

Researchers grow stem cells in a lab. These stem cells are manipulated to specialize into specific types of cells, such as heart muscle cells, blood cells or nerve cells.

The specialized cells can then be implanted into a person. For example, if the person has heart disease, the cells could be injected into the heart muscle. The healthy transplanted heart muscle cells could then contribute to repairing the injured heart muscle.

Researchers have already shown that adult bone marrow cells guided to become heart-like cells can repair heart tissue in people, and more research is ongoing.

Have stem cells already been used to treat diseases?

Yes. Doctors have performed stem cell transplants, also known as bone marrow transplants, for many decades. In hematopoietic stem cell transplants, stem cells replace cells damaged by chemotherapy or disease or serve as a way for the donor's immune system to fight some types of cancer and blood-related diseases. Leukemia, lymphoma, neuroblastoma and multiple myeloma often are treated this way. These transplants use adult stem cells or umbilical cord blood.

Researchers are testing adult stem cells to treat other conditions, including some degenerative diseases such as heart failure.

What are the potential problems with using embryonic stem cells in humans?

For embryonic stem cells to be useful, researchers must be certain that the stem cells will differentiate into the specific cell types desired.

Researchers have discovered ways to direct stem cells to become specific types of cells, such as directing embryonic stem cells to become heart cells. Research is ongoing in this area.

Embryonic stem cells also can grow irregularly or specialize in different cell types spontaneously. Researchers are studying how to control the growth and development of embryonic stem cells.

Embryonic stem cells also might trigger an immune response in which the recipient's body attacks the stem cells as foreign invaders, or the stem cells might simply fail to function as expected, with unknown consequences. Researchers continue to study how to avoid these possible complications.

What is therapeutic cloning, and what benefits might it offer?

Therapeutic cloning, also called somatic cell nuclear transfer, is a way to create versatile stem cells independent of fertilized eggs. In this technique, the nucleus is removed from an unfertilized egg. This nucleus contains the genetic material. The nucleus also is removed from the cell of a donor.

This donor nucleus is then injected into the egg, replacing the nucleus that was removed, in a process called nuclear transfer. The egg is allowed to divide and soon forms a blastocyst. This process creates a line of stem cells that is genetically identical to the donor's cells — in essence, a clone.

Some researchers believe that stem cells derived from therapeutic cloning may offer benefits over those from fertilized eggs because cloned cells are less likely to be rejected once transplanted back into the donor. And it may allow researchers to see exactly how a disease develops.

Has therapeutic cloning in people been successful?

No. Researchers haven't been able to successfully perform therapeutic cloning with humans despite success in a number of other species.

Researchers continue to study the potential of therapeutic cloning in people.

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• Stem cell basics. National Institutes of Health. https://stemcells.nih.gov/info/basics/stc-basics/#stc-I. Accessed March 21, 2024.
• Lovell-Badge R, et al. ISSCR guidelines for stem cell research and clinical translation: The 2021 update. Stem Cell Reports. 2021; doi:10.1016/j.stemcr.2021.05.012.
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• NIH guidelines for human stem cell research. National Institutes of Health. https://stemcells.nih.gov/research-policy/guidelines-for-human-stem-cell-research. Accessed March 21, 2024.
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• Alessandrini M, et al. Stem cell therapy for neurological disorders. South African Medical Journal. 2019; doi:10.7196/SAMJ.2019.v109i8b.14009.
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Current state of stem cell-based therapies: an overview

Riham mohamed aly.

1 Department of Basic Dental Science, National Research Centre, Cairo, Egypt;

2 Stem Cell Laboratory, Center of Excellence for Advanced Sciences, National Research Centre, Cairo, Egypt

Recent research reporting successful translation of stem cell therapies to patients have enriched the hope that such regenerative strategies may one day become a treatment for a wide range of vexing diseases. In fact, the past few years witnessed, a rather exponential advancement in clinical trials revolving around stem cell-based therapies. Some of these trials resulted in remarkable impact on various diseases. In this review, the advances and challenges for the development of stem-cell-based therapies are described, with focus on the use of stem cells in dentistry in addition to the advances reached in regenerative treatment modalities in several diseases. The limitations of these treatments and ongoing challenges in the field are also discussed while shedding light on the ethical and regulatory challenges in translating autologous stem cell-based interventions, into safe and effective therapies.

Introduction

Cell-based therapy as a modality of regenerative medicine is considered one of the most promising disciplines in the fields of modern science & medicine. Such an advanced technology offers endless possibilities for transformative and potentially curative treatments for some of humanities most life threatening diseases. Regenerative medicine is rapidly becoming the next big thing in health care with the particular aim of repairing and possibly replacing diseased cells, tissues or organs and eventually retrieving normal function. Fortunately, the prospect of regenerative medicine as an alternative to conventional drug-based therapies is becoming a tangible reality by the day owing to the vigorous commitment of the research communities in studying the potential applications across a wide range of diseases like neurodegenerative diseases and diabetes, among many others ( 1 ).

Recent research reporting successful translation of stem cell therapies to patients have enriched the hope that such regenerative strategies may one day become a treatment for a wide range of vexing diseases ( 2 ). In fact, the past few years witnessed, a rather exponential advancement in clinical trials revolving around stem cell-based therapies. Some of these trials resulted in remarkable impact on various diseases ( 3 ). For example, a case of Epidermolysis Bullosa manifested signs of skin recovery after treatment with keratinocyte cultures of epidermal stem cells ( 4 ). Also, a major improvement in eyesight of patients suffering from macular degeneration was reported after transplantation of patient-derived induced pluripotent stem cells (iPSCs) that were induced to differentiate into pigment epithelial cells of the retina ( 5 ).

However, in spite of the increased amount of publications reporting successful cases of stem cell-based therapies, a major number of clinical trials have not yet acquired full regulatory approvals for validation as stem cell therapies. To date, the most established stem cell treatment is bone marrow transplants to treat blood and immune system disorders ( 1 , 6 , 7 ).

In this review, the advances and challenges for the development of stem-cell-based therapies are described, with focus on the use of stem cells in dentistry in addition to the advances reached in regenerative treatment modalities in several diseases. The limitations of these treatments and ongoing challenges in the field are also discussed while shedding light on the ethical and regulatory challenges in translating autologous stem cell-based interventions, into safe and effective therapies.

Stem cell-based therapies

Stem cell-based therapies are defined as any treatment for a disease or a medical condition that fundamentally involves the use of any type of viable human stem cells including embryonic stem cells (ESCs), iPSCs and adult stem cells for autologous and allogeneic therapies ( 8 ). Stem cells offer the perfect solution when there is a need for tissue and organ transplantation through their ability to differentiate into the specific cell types that are required for repair of diseased tissues.

However, the complexity of stem cell-based therapies often leads researchers to search for stable, safe and easily accessible stem cells source that has the potential to differentiate into several lineages. Thus, it is of utmost importance to carefully select the type of stem cells that is suitable for clinical application ( 7 , 9 ).

Stem cells hierarchy

There are mainly three types of stem cells. All three of them share the significant property of self-renewal in addition to a unique ability to differentiate. However, it should be noted that stem cells are not homogeneous, but rather exist in a developmental hierarchy ( 10 ). The most basic and undeveloped of stem cells are the totipotent stem cells. These cells are capable of developing into a complete embryo while forming the extra-embryonic tissue at the same time. This unique property is brief and starts with the fertilization of the ovum and ends when the embryo reaches the four to eight cells stage. Following that cells undergo subsequent divisions until reaching the blastocyst stage where they lose their totipotency property and assume a pluripotent identity where cells are only capable of differentiating into every embryonic germ layer (ectoderm, mesoderm and endoderm). Cells of this stage are termed “embryonic stem cells” and are obtained by isolation from the inner cell mass of the blastocyst in a process that involves the destruction of the forming embryo. After consecutive divisions, the property of pluripotency is lost and the differentiation capability becomes more lineage restricted where the cells become multipotent meaning that they can only differentiate into limited types of cells related to the tissue of origin. This is the property of “adult stem cells”, which helps create a state of homeostasis throughout the lifetime of the organism. Adult stem cells are present in a metabolically quiescent state in almost all specialized tissues of the body, which includes bone marrow and oral and dental tissues among many others ( 11 ).

Many authors consider adult stem cells the gold standard in stem cell-based therapies ( 12 , 13 ). Adult stem cells demonstrated signs of clinical success especially in hematopoietic transplants ( 14 , 15 ). In contrast to ESCs, adult stem cells are not subjected to controversial views regarding their origin. The fact that ESCs derivation involves destruction of human embryos renders them unacceptable for a significant proportion of the population for ethical and religious convictions ( 16 - 18 ).

Turning point in stem cell research

It was in 2006 when Shinya Yamanka achieved a scientific breakthrough in stem cell research by succeeding in generating cells that have the same properties and genetic profile of ESCs. This was achieved via the transient over-expression of a cocktail of four transcription factors; OCT4, SOX2, KLF4 and MYC in, fully differentiated somatic cells, namely fibroblasts ( 19 , 20 ). These cells were called iPSCs and has transformed the field of stem cell research ever since ( 21 ). The most important feature of these cells is their ability to differentiate into any of the germ layers just like ESCs precluding the ethical debate surrounding their use. The development of iPSCs technology has created an innovative way to both identify and treat diseases. Since they can be generated from the patient’s own cells, iPSCs thus present a promising potential for the production of pluripotent derived patient-matched cells that could be used for autologous transplantation. True these cells symbolize a paradigm shift since they enable researchers to directly observe and treat relevant patient cells; nevertheless, a number of challenges still need to be addressed before iPSCs-derived cells can be applied in cell therapies. Such challenges include; the detection and removal of incompletely differentiated cells, addressing the genomic and epigenetic alterations in the generated cells and overcoming the tumorigenicity of these cells that could arise on transplantation ( 22 ).

Therapeutic translation of stem cell research

With the rapid increase witnessed in stem cell basic research over the past years, the relatively new research discipline “Translational Research” has evolved significantly building up on the outcomes of basic research in order to develop new therapies. The clinical translation pathway starts after acquiring the suitable regulatory approvals. The importance of translational research lies in it’s a role as a filter to ensure that only safe and effective therapies reach the clinic ( 23 ). It bridges the gap from bench to bed. Currently, some stem cell-based therapies utilizing adult stem cells are clinically available and mainly include bone marrow transplants of hematopoietic stem cells and skin grafts for severe burns ( 23 ). To date, there are more than 3,000 trials involving the use of adult stem cells registered in WHO International Clinical Trials Registry. Additionally, initial trials involving the new and appealing iPSCs based therapies are also registered. In fact, the first clinical attempt employing iPSCs reported successful results in treating macular degeneration ( 24 ). Given the relative immaturity in the field of cellular therapy, the outcomes of such trials shall facilitate the understanding of the timeframes needed to achieve successful therapies and help in better understanding of the diseases. However, it is noteworthy that evaluation of stem cell-based therapies is not an easy task since transplantation of cells is ectopic and may result in tumor formation and other complications. This accounts for the variations in the results reported from previous reports. The following section discusses the published data of some of the most important clinical trials involving the use of different types of stem cells both in medicine and in dentistry.

Stem cell-based therapy for neurodegenerative diseases

The successful generation of neural cells from stem cells in vitro paved the way for the current stem cell-based clinical trials targeting neurodegenerative diseases ( 25 , 26 ). These therapies do not just target detaining the progression of irrecoverable neuro-degenerative diseases like Parkinson’s, Alzheimer’s, amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS), but are also focused on completely treating such disorders.

Parkinson’s disease (PD)

PD is characterized by a rapid loss of midbrain dopaminergic neurons. The first attempt for using human ESC cells to treat PD was via the generation of dopaminergic-like neurons, later human iPSCs was proposed as an alternative to overcome ESCs controversies ( 27 ). Both cells presented hope for obtaining an endless source of dopaminergic neurons instead of the previously used fetal brain tissues. Subsequently, protocols that mimicked the development of dopaminergic neurons succeeded in generating dopaminergic neurons similar to that of the midbrain which were able to survive, integrate and functionally mature in animal models of PD preclinically ( 28 ). Based on the research presented by different groups; the “Parkinson’s Global Force” was formed which aimed at guiding researchers to optimize their cell characterization and help promote the clinical progress toward successful therapy. Recently, In August 2018, Shinya Yamanka initiated the first approved clinical trial to treat PD using iPSCs. Seven patients suffering from moderate PD were recruited ( 29 ). Donor matched allogeneic cells were used to avoid any genetic influence of the disease. The strategy behind the trial involved the generation of dopaminergic progenitors followed by surgical transplantation into the brains of patients by a special device. In addition, immunosuppressant medications were given to avoid any adverse reaction. Preliminary results so far revealed the safety of the treatment.

MS is an inflammatory and neurodegenerative autoimmune disease of the central nervous system. Stem cell-based therapies are now exploring the possibility of halting the disease progression and reverse the neural damage. A registered phase 1 clinical trial was conducted by the company Celgene TM in 2014 using placental-derived mesenchymal stem cells (MSCs) infusion to treat patients suffering from MS ( 30 ). This trial was performed at 6 centers in the United States and 2 centers in Canada and included 16 patients. Results demonstrated that cellular infusions were safe with no signs of paradoxical aggravation. However, clinical responses from patients indicated that the cellular treatment did not improve the MS condition ( 31 ). For the last decade immunoablative therapy demonstrated accumulative evidence of inducing long-term remission and improvement of disability caused by MS. This approach involves the replacement of the diseased immune system through administration of high-dose immunosuppressive therapy followed by hematopoietic stem cells infusion ( 32 ). However, immunoablation strategies demonstrated several complications such as infertility and neurological disabilities. A number of randomized controlled trials are planned to address these concerns ( 32 ). Currently, new and innovative stem cell-based therapies for MS are only in the initial stages, and are based on different mechanisms exploring the possibility of replacing damaged neuronal tissue with neural cells derived from iPSCs however, the therapeutic potential of iPSCs is still under research ( 33 ).

ALS is a neurodegenerative disease that causes degeneration of the motor neurons which results in disturbance in muscle performance. The first attempt to treat ALS was through the transplantation of MSCs in a mouse model. The outcomes of this experiment were promising and resulted in a decrease of the disease manifestations and thus providing proof of principal ( 34 ). Based on these results, several planned/ongoing clinical trials are on the way. These trials mainly assess the safety of the proposed concept and have not proved clinical success to date. Notably, while pre-clinical studies have reported that cells derived from un-diseased individuals are superior to cells from ALS patients; most of the clinical trials attempted have employed autologous transplantation. This information may account for the absence of therapeutic improvement reported ( 35 ).

Spinal cord injury

Other neurologic indications for the use of stem cells are spinal cord injuries. Though the transplantation of different forms of neural stem cells and oligo-dendrocyte progenitors has led to growth in the axons in addition to neural connectivity which presents a possibility for repair ( 36 ), proof of recovered function has yet to be established in stringent clinical trials. Nevertheless, Japan has recently given approval to stem-cell treatment for spinal-cord injuries. This approval was based on clinical trials that are yet to be published and involves 13 patients, who are suffering from recent spinal-cord injury. The Japanese team discovered that injection of stem cells isolated from the patients’ bone marrow aided in regaining some lost sensation and mobility. This is the first stem cell-based therapy targeting spinal-cord injuries to gain governmental approval to offer to patients ( 37 ).

Stem cell-based therapies for ocular diseases

A huge number of the currently registered clinical trials for stem cell-based therapies target ocular diseases. This is mainly due to the fact that the eye is an immune privileged site. Most of these trials span various countries including Japan, China, Israel, Korea, UK, and USA and implement allogeneic ESC lines ( 35 , 36 ). Notably, the first clinical trial to implement the use autologous iPSCs-derived retinal cells was in Japan which followed the new regulatory laws issued in 2014 by Japan’s government to regulate regenerative medicine applications. Two patients were recruited in this trial, the first one received treatment for macular degeneration using iPSCs-generated retinal cell sheet ( 37 ). After 1 year of follow-up, there were no signs of serious complications including abnormal proliferation and systemic malignancy. Moreover, there were no signs of rejection of the transplanted retinal epithelial sheet in the second year follow-up. Most importantly, the signs of corrected visual acuity of the treated eye were reported. These results were enough to conclude that iPSCs-based autologous transplantation was safe and feasible ( 38 ). It is worthy to mention that the second patient was withdrawn from the study due to detectable genetic variations the patient’s iPSCs lines which was not originally present in the patient’s original fibroblasts. Such alterations may jeopardize the overall safety of the treatment. The fact that this decision was taken, even though the performed safety assays did not demonstrate tumorgenicity in the iPSCs-derived retinal pigment epithelium (RPE) cells, indicates that researchers in the field of iPSCs have full awareness of the importance of safety issues ( 39 ).

Stem cell-based therapies for treatment of diabetes

Pancreatic beta cells are destructed in type 1 diabetes mellitus, because of disorders in the immune system while in type 2 insulin insufficiency is caused by failure of the beta-cell to normally produce insulin. In both cases the affected cell is the beta cell, and since the pancreas does not efficiently regenerate islets from endogenous adult stem cells, other cell sources were tested ( 38 ). Pluripotent stem cells (PSCs) are considered the cells of choice for beta cell replacement strategies ( 39 ). Currently, there are a few industry-sponsored clinical trials that are registered targeting beta cell replacement using ESCs. These trials revolve around the engraftment of insulin-producing beta cells in an encapsulating device subcutaneously to protect the cells from autoimmunity in patients with type 1 diabetes ( 40 ). The company ViaCyte TM in California recently initiated a phase I/II trial ( {"type":"clinical-trial","attrs":{"text":"NCT02239354","term_id":"NCT02239354"}} NCT02239354 ) in 2014 in collaboration with Harvard University. This trial involves 40 patients and employs two subcutaneous capsules of insulin producing beta cells generated from ESCs. The results shall be interesting due to the ease of monitoring and recovery of the transplanted cells. The preclinical studies preceding this trial demonstrated successful glycemic correction and the devices were successfully retrieved after 174 days and contained viable insulin-producing cells ( 41 ).

Stem cells in dentistry

Stem cells have been successfully isolated from human teeth and were studied to test their ability to regenerate dental structures and periodontal tissues. MSCs were reported to be successfully isolated from dental tissues like dental pulp of permanent and deciduous teeth, periodontal ligament, apical papilla and dental follicle ( 42 - 44 ). These cells were described as an excellent cell source owing to their ease of accessibility, their ability to differentiate into osteoblasts and odontoblasts and lack of ethical controversies ( 45 ). Moreover, dental stem cells demonstrated superior abilities in immunomodulation properties either through cell to cell interaction or via a paracrine effect ( 46 ). Stem cells of non-dental origin were also suggested for dental tissue and bone regeneration. Different approaches were investigated for achieving dental and periodontal regeneration ( 47 ); however, assessments of stem cells after transplantation still require extensive studying. Clinical trials have only recently begun and their results are yet to be fully evaluated. However, by carefully applying the knowledge acquired from the extensive basic research in dental and periodontal regeneration, stem cell-based dental and periodontal regeneration may soon be a readily available treatment. To date, there are more than 6,000 clinical trials involving the use of with stem cells, however only a total of 44 registered clinical trials address oral diseases worldwide ( 48 ). Stem cell-based clinical trials with reported results targeting the treatment of oral disease are discussed below.

Dental pulp regeneration

The first human clinical study using autologous dental pulp stem cells (DPSCs) for complete pulp regeneration was reported by Nakashima et al. in 2017 ( 49 ). This pilot study was based on extensive preclinical studies conducted by the same group ( 50 ). Patients with irreversible pulpitis were recruited and followed up for 6 months following DPSCs’ transplantation. Granulocyte colony-stimulating factor was administered to induce stem cell mobilization to enrich the stem cell populations. The research team reported that the use of DPSCs seeded on collagen scaffold in molars and premolars undergoing pulpectomy was safe. No adverse events or toxicity were demonstrated in the clinical and laboratory evaluations. Positive electric pulp testing was obtained after cell transplantation in all patients. Moreover, magnetic resonance imaging of the de - novo tissues formed in the root canal demonstrated similar results to normal pulp, which indicated successful pulp regeneration. A different group conducted a clinical trial that recruited patients diagnosed with necrotic pulp. Autologous stem cells from deciduous teeth were employed to induce pulp regeneration ( 51 ). Follow-up of the cases after a year from the intervention reported evidence of pulp regeneration with vascular supply and innervation. In addition, no signs of adverse effects were observed in patients receiving DPSCs transplantation. Both trials are proceeding with the next phases, however the results obtained are promising.

Periodontal tissue regeneration

Aimetti et al. performed a study which included eleven patients suffering from chronic periodontitis and have one deep intra bony defect in addition to the presence of one vital tooth that needs extraction ( 52 ). Pulp tissue was passed through 50-µm filters in presence of collagen sponge scaffold and was followed by transplantation in the bony defects caused by periodontal disease. Both clinical and radiographic evaluations confirmed the efficacy of this therapeutic intervention. Periodontal examination, attachment level, and probe depth showed improved results in addition to significant stability of the gingival margin. Moreover, radiographic analysis demonstrated bone regeneration.

Regeneration of mandibular bony defects

The first clinical study using DPSCs for oro-maxillo-facial bone regeneration was conducted in 2009 ( 53 ). Patients in this study suffered from extreme bone loss following extraction of third molars. A bio-complex composed of DPSCs cultured on collagen sponge scaffolds was applied to the affected sites. Vertical repair of the damaged area with complete restoration of the periodontal tissue was demonstrated six months after the treatment. Three years later, the same group published a report evaluating the stability and quality of the regenerated bone after DPSCs transplantation ( 54 ). Histological and advanced holotomography demonstrated that newly formed bone was uniformly vascularized. However, it was of compact type, rather than a cancellous type which is usually the type of bone in this region.

Stem cells for treatment of Sjögren’s syndrome

Sjögren’s syndrome (SS) is a systemic autoimmune disease marked by dry mouth and eyes. A novel therapeutic approach for SS. utilizing the infusion of MSCs in 24 patients was reported by Xu et al. in 2012 ( 55 ). The strategy behind this treatment was based on the immunologic regulatory functions of MSCs. Infused MSCs migrated toward the inflammatory sites in a stromal cell-derived factor-1-dependent manner. Results reported from this clinical trial demonstrated suppressed autoimmunity with subsequent restoration of salivary gland secretion in SS patients.

Stem cells and tissue banks

The ability to bank autologous stem cells at their most potent state for later use is an essential adjuvant to stem cell-based therapies. In order to be considered valid, any novel stem cell-based therapy should be as effective as the routine treatment. Thus, when appraising a type of stem cells for application in cellular therapies, issues like immune rejection must be avoided and at the same time large numbers of stem cells must be readily available before clinical implementation. iPSCs theoretically possess the ability to proliferate unlimitedly which pose them as an attractive source for use in cell-based therapies. Unlike, adult stem cells iPSCs ability to propagate does not decrease with time ( 22 ). Recently, California Institute for Regenerative Medicine (CIRM) has inaugurated an iPSCs repository to provide researchers with versatile iPSCs cell lines in order to accelerate stem cell treatments through studying genetic variation and disease modeling. Another important source for stem cells banking is the umbilical cord. Umbilical cord is immediately cryopreserved after birth; which permits stem cells to be successfully stored and ready for use in cell-based therapies for incurable diseases of a given individuals. However, stem cells of human exfoliated deciduous teeth (SHEDs) are more attractive as a source for stem cell banking. These cells have the capacity to differentiate into further cell types than the rest of the adult stem cells ( 56 ). Moreover, procedures involving the isolation and cryopreservation of these cells are un-complicated and not aggressive. The most important advantage of banking SHEDs is the insured autologous transplant which avoids the possibility of immune rejection ( 57 ). Contrary to cord blood stem cells, SHEDs have the ability to differentiate into connective tissues, neural and dental tissues ( 58 ) Finally, the ultimate goal of stem cell banking, is to establish a repository of high-quality stem cell lines derived from many individuals for future use in therapy.

Current regulatory guidelines for stem cell-based therapies

With the increased number of clinical trials employing stem cells as therapeutic approaches, the need for developing regulatory guidelines and standards to ensure patients safety is becoming more and more essential. However, the fact that stem cell therapy is rather a new domain makes it subject to scientific, ethical and legal controversies that are yet to be regulated. Leading countries in the field have devised guidelines serving that purpose. Recently, the Food and Drug Administration (FDA) has released regulatory guidelines to ensure that these treatments are safe and effective ( 59 ). These guidelines state that; treatments involving stem cells that have been minimally manipulated and are intended for homogeneous use do not require premarket approval to come into action and shall only be subjected to regulatory guidelines against disease transmission. In 2014, a radical regulatory reform in Japan occurred with the passing of two new laws that permitted conditional approval of cell-based treatments following early phase clinical trials on the condition that clinical safety data are provided from at least ten patients. These laws allow skipping most of the traditional criteria of clinical trials in what was described as “fast track approvals” and treatments were classified according to risk ( 60 ). To date, the treatments that acquired conditional approval include those targeting; spinal-cord injury, cardiac disease and limb ischemia ( 61 ). Finally, regulatory authorities are now demanding application of standardization and safety regulations protocols for cellular products, which include the use of Xeno-free culture media, recombinant growth factors in addition to “Good Manufacturing Practice” (GMP) culture supplies.

Challenges & ethical issues facing stem cell-based therapies

Stem cell-based therapies face many obstacles that need to be urgently addressed. The most persistent concern is the ethical conflict regarding the use of ESCs. As previously mentioned, ESCs are far superior regarding their potency; however, their derivation requires destruction human embryos. True, the discovery of iPSCs overcame this concern; nevertheless, iPSCs themselves currently face another ethical controversy of their own which addresses their unlimited capacity of differentiation with concerns that these cells could one day be applied in human cloning. The use of iPSCs in therapy is still considered a high-risk treatment modality, since transplantation of these cells could induce tumor formation. Such challenge is currently addressed through developing optimized protocols to ensure their safety in addition to developing global clinical-grade iPSCs cell lines before these cells are available for clinical use ( 61 ). As for MSCs, these cells have been universally considered safe, however continuous monitoring and prolonged follow-up should be the focus of future research to avoid the possibility of tumor formation after treatments ( 62 ). Finally, it could be postulated that one of the most challenging ethical issues faced in the field of stem cell-based therapies at the moment, is the increasing number of clinics offering unproven stem cell-based treatments. Researchers are thus morally obligated to ensure that ethical considerations are not undermined in pursuit of progress in clinical translation.

Conclusions

Stem cell therapy is becoming a tangible reality by the day, thanks to the mounting research conducted over the past decade. With every research conducted the possibilities of stem cells applications increased in spite of the many challenges faced. Currently, progress in the field of stem cells is very promising with reports of clinical success in treating various diseases like; neurodegenerative diseases and macular degeneration progressing rapidly. iPSCs are conquering the field of stem cells research with endless possibilities of treating diseases using patients own cells. Regeneration of dental and periodontal tissues using MSCs has made its way to the clinic and soon enough will become a valid treatment. Although, challenges might seem daunting, stem cell research is advancing rapidly and cellular therapeutics is soon to be applicable. Fortunately, there are currently tremendous efforts exerted globally towards setting up regulatory guidelines and standards to ensure patients safety. In the near future, stem cell-based therapies shall significantly impact human health.

Acknowledgments

Funding: None.

Ethical Statement: The author is accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/ .

Conflicts of Interest: The author has completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/sci-2020-001 ). The author has no conflicts of interest to declare.

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Researchers at the IBBS are studying stem cells to figure out how they can give rise to a complex body, how cells of the body can revert back to stem cells and how this knowledge can be used to develop therapies for diseases and injuries.

Stem cells are cells that don’t have an identity but have the potential to develop into many types of cells for many purposes, liking building a complete organism, healing a wound or replacing old, worn-out cells in a tissue.

Embryonic stem cells can become any of the cells in the body and can form entire animals.

Not all stem cells come from embryos, adult stem cells are found throughout the body too. These cells don’t have the ability to become any cell in the body, but can transform into many different cell types. For instance, there are stem cells in our bone marrow that can become fat cells, cartilage cells or bone cells, but they can’t become eye cells or skin cells. Researchers have also figured out how to make adult cells, like a skin cell, turn back into cells with the properties of embryonic stem cells, called induced pluripotent stem cells or iPS cells for short.

Erika Matunis ,  in the Department of Cell Biology , studies in fruit flies how testis stem cells decide to stay stem cells and not become other cell types, like sperm. She has also discovered how cells that are turning into other cell types can revert back to stem cells if the permanent reservoir of stem cells is depleted and she is exploring the mechanism of how this happens. Her research, learning more about the most fundamental aspects of stem cell biology, helps all stem cell researchers better understand the cells they work with.

Jennifer Elisseeff ,  of the Department of Biomedical Engineering , studies the differences between embryonic stem cells and adult stem cells. She has found that embryonic stem cells are better at forming new tissues, whereas adult stem cells are better at secreting therapeutic molecules that promote healing of damaged tissue. Elisseeff is particularly interested in the factors released by stem cells that can help a tissue heal. She uses this information in the development of biosynthetic (part-natural and part-man-made) materials used for therapies. One of the materials her lab has developed is a bio-adhesive—essentially a glue that can be used in the body that is made of part synthetic and part natural components. The glue is used in conjunction with stitches to help prevent leakage of blood or fluids, but it’s flexible enough to allow cells to move in and heal the incision. Also, Elisseeff is collaborating with the military to develop a treatment for soft tissue facial reconstruction for people who have suffered severe trauma. They are developing tissue blueprints that can be transplanted in the face—or any other place in the body for that matter— that would allow a person’s own cells to move into a region to heal and restructure the tissue.

Warren Grayson ,  of the Department of Biomedical Engineering , takes stem cells from fat and bone marrow as well as stem cells that have the potential to become many different cell types, known as pluripotent stem cells, and coaxes them to regenerate bone or skeletal muscle in the lab. He does this by incubating stem cells in biosynthetic structures to give the cells a structured three-dimensional volume to grow in, and then places these either in bioreactors that provide heat, nutrients, movement, mechanical stress or control of any other condition like oxygen concentration to guide the stem cell to become a specific cell type or within a defect in animals to study the regenerative process. He hopes to one day be able to take a person’s own stem cells and grow tissues, like bone or muscle, to be implanted into their body to replace damaged tissue. Using a person’s own cells and tissues will reduce the likelihood that the transplanted tissue will be rejected by the immune system.

Related Links :  Stem Cell Research at Johns Hopkins

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• Review Article
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• Published: 02 October 2023

A new era of stem cell and developmental biology: from blastoids to synthetic embryos and beyond

• Yunhee Kim   ORCID: orcid.org/0000-0001-5379-7744 1 , 2   na1 ,
• Inha Kim   ORCID: orcid.org/0009-0007-3263-4541 1 , 2   na1 &
• Kunyoo Shin   ORCID: orcid.org/0000-0002-1519-9839 1 , 2  

Experimental & Molecular Medicine volume  55 ,  pages 2127–2137 ( 2023 ) Cite this article

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• Disease model
• Embryogenesis
• Organogenesis
• Pattern formation

Recent discoveries in stem cell and developmental biology have introduced a new era marked by the generation of in vitro models that recapitulate early mammalian development, providing unprecedented opportunities for extensive research in embryogenesis. Here, we present an overview of current techniques that model early mammalian embryogenesis, specifically noting models created from stem cells derived from two significant species: Homo sapiens , for its high relevance, and Mus musculus , a historically common and technically advanced model organism. We aim to provide a holistic understanding of these in vitro models by tracing the historical background of the progress made in stem cell biology and discussing the fundamental underlying principles. At each developmental stage, we present corresponding in vitro models that recapitulate the in vivo embryo and further discuss how these models may be used to model diseases. Through a discussion of these models as well as their potential applications and future challenges, we hope to demonstrate how these innovative advances in stem cell research may be further developed to actualize a model to be used in clinical practice.

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Introduction.

The 1995 Nobel Prize in Physiology or Medicine, awarded to Edward B. Lewis, Christiane Nüsslein-Volhard, and Eric F. Wieschaus, marked a turning point in the field of modern developmental biology. By introducing mutations in Drosophila embryos, these researchers found key genes associated with regulating several significant early developmental processes, such as segmentation and polarization in Drosophila . Their groundbreaking discoveries in the genetic control of embryonic development laid the foundation for a deeper understanding of the molecular mechanisms governing the formation of multicellular organisms 1 , 2 .

Over the past three decades, substantial progress has been made in developmental biology ever since the foundation of several techniques, such as genetic engineering techniques, wherein various animal model systems were actively utilized to further enhance the understanding of genetic functions within the processes of embryonic development. However, studying human development has remained a significant challenge due to the inherent differences between conventional model organisms and humans 3 , ethical concerns about using human embryos 4 , and physical inaccessibility due to the intrauterine development of in vivo human embryos.

Recently, the rapid advancement of stem cell research has enabled the generation of stem cell-derived human organoids 5 , which are three-dimensional, self-organizing structures that mimic various human organs. This remarkable progress has expanded the boundaries of human biology research, allowing the investigation of human biological processes in a more physiologically relevant context 6 . In addition to organoids, recent advances in stem cell research have even facilitated the development of early embryonic models that mimic human embryos. Early embryonic models provide an opportunity to investigate molecular mechanisms that regulate human embryogenesis with better scalability and in a relatively ethical manner, thereby overcoming the major limitations of using natural human embryos 7 , 8 , 9 , 10 .

Early embryonic models were generated by using embryonic stem cells (ESCs) 11 , which possess the ability to self-renew and differentiate into a variety of specific cell types of the body. The potency of ESCs varies depending on their epigenetic traits and culture environment 12 . These shifts in potency allow the induction of various in vitro models, including synthetic whole embryo models that incorporate all significant cell types, such as those derived from both the embryo and extraembryonic tissues 8 . This review describes the process by which stem cells, including ESCs in relation to their potency, are utilized to create these in vitro models.

This review aims to explore developments in stem cell research, focusing on stem cell-based in vitro early embryonic developmental models. Overall, this review article aims to provide a comprehensive analysis of the new era of stem cell research, examining the potential uses of developmental-stage-specific embryo models for disease modeling and possible clinical applications. By providing a thorough discussion of these technologies, we hope to inspire further research and advancements in the field.

Embryonic stem cells (ESCs): the building blocks of synthetic embryos

Stem cells are self-renewing cells with the potential to differentiate into a wide variety of cell types 13 . Based on their origin, they are divided into two major categories: ESCs and adult stem cells (AdSCs). ESCs are derived from the inner cell mass (ICM) of blastocysts and can differentiate into any type of cell in the body 14 , whereas AdSCs are found in various tissues in our body and help maintain tissue homeostasis but can differentiate into only a limited number of cell types relative to ESCs 13 , 15 . Early embryonic in vitro models were all derived from ESCs or induced pluripotent stem cells (iPSCs) 16 of equivalent potency. This section focuses on ESCs, which serve as the foundation for creating in vitro models, and analyzes their diverse potencies (Fig. 1a ).

a Timeline of significant events in stem cell research facilitating the generation of synthetic embryos. b Illustration of early mammalian development in vivo (left), as well as the in vitro counterparts—naïve and primed ES cells—represented as cell states (right). Each developmental stage of the mammalian embryo is depicted from the fertilization of the egg (Day 0) to the implantation of the blastocyst (Day 9). Pre- (top) and post-implantation (bottom) stem cells derived in vitro are listed in the middle, as well as the original research in which they were discovered. The ESCs that correspond to the pre- and post-implantation states are naïve (top) and primed (bottom) ESCs, respectively, and the degrees of the states are represented on the right (ascending, more naïve; descending, more primed). Morphogens that regulate their states are written in between.

Birth of ESCs

The concept of ESCs was first proposed in the 1950s by Stevens and Little, who discovered that spontaneous testicular teratomas in an inbred mouse strain originated from primordial germ cells and that these cells can differentiate into a variety of cell types 17 . This discovery illuminated the pluripotency of germ cells, opening the way for further investigation into the origin and differentiation potential of other stem cell types. Kleinsmith and Pierce expanded upon these findings by demonstrating the multipotentiality of single embryonal carcinoma cells 18 .

The development of in vitro culture methods also played a crucial role in the establishment of ESCs. Evans pioneered the use of irradiated chick embryo fibroblasts as feeder cells to culture mammalian stem cells in vitro, which enabled the maintenance and expansion of pluripotent stem cells (PSCs) 19 . Based on these findings, Evans, Kaufman, and Martin successfully derived mouse ESCs (mESCs) from the ICM of blastocysts, marking the beginning of modern stem cell research 20 , 21 . Building on the advancements in mESC research, the scientific focus shifted to human ESCs (hESCs), leading to a significant breakthrough in 1998 when Thomson et al. successfully derived and cultured hESCs in vitro by isolating them from the ICM of human blastocysts 14 . Both mouse and human ESCs possess the capacity for self-renewal and pluripotency, which allows them to maintain an undifferentiated state while proliferating in culture and to differentiate into any cell type of the three germ layers (endoderm, mesoderm, and ectoderm) 14 , 20 .

Although initially established mESCs and hESCs share certain traits, they also display distinct differences in morphological, transcriptional, and epigenetic features 22 . mESCs, for instance, have a tendency to form a dome-like structure, whereas hESCs typically exhibit a flattened shape 22 . Both mESCs and hESCs express the pluripotency-related transcription factor octamer-binding transcription factor 4 (OCT4), albeit with distinct regulatory mechanisms. In mESCs, OCT4 expression is regulated by its distal enhancer, while in hESCs, its expression is mainly regulated by its proximal enhancer 23 , 24 .

As subsequent research progressed, a method was developed to culture mESCs that exhibited characteristics similar to hESCs 25 , 26 . These cells, derived from the epiblast of post-implantation embryos, were termed mouse epiblast stem cells (mEpiSCs) and shared morphology and a variety of epigenetic properties similar to hESCs 26 . Continuous investigations have revealed that the differences between mESCs and hESCs originate from the fact that these ESCs represent different stages of development, thereby influencing their slightly different differentiation potentials.

Naïve and primed pluripotent states of ESCs

Mammalian ESCs are known to exist in a continuum of configurations, which at either end of the spectrum lie two different pluripotent states: naïve and primed 12 . These states have distinct molecular and functional characteristics, and understanding their differences is crucial for optimizing the use of ESCs in modeling early embryos in vitro.

Naïve ESCs, compared to primed ESCs, correspond to an earlier developmental stage of pluripotent cells. They are derived from the pre-implantation ICM during the early blastocyst stage 27 (Fig. 1b ). These cells exhibit characteristics such as having a relatively unrestricted differentiation potential, allowing them to differentiate into both embryonic (epiblast) and extraembryonic tissue (hypoblast). In addition, they have a more open chromatin structure on developmental regulatory gene promoters, accompanied by a global reduction in DNA methylation 27 , 28 .

In contrast, primed ESCs are derived from a developmentally more advanced stage, specifically from post-implantation epiblast cells 27 (Fig. 1b ). These cells exhibit a relatively restricted differentiation potential; they predominantly contribute to embryonic components, unlike their naïve counterparts 25 . This restriction is likely due to their relatively closed chromatin structure on lineage regulatory genes 29 .

The first mESCs established were in the naïve state 20 , 21 , while the initial hESCs and mEpiSCs that were established resembled the primed state of pluripotency 14 , 25 . Over several years, efforts to establish human naïve ESCs have led researchers to gain insights into the molecular mechanisms and signaling pathways regulating pluripotency. These key insights included the identification of factors essential for maintaining naïve pluripotency in mESCs, such as leukemia inhibitory factor (LIF), which activates the Janus kinase-signal transducer and activator of transcription 3 (JAK-STAT3) signaling pathway 30 , 31 . The development of specific culture conditions to derive and maintain mEpiSCs facilitated further investigation into the signaling pathways that regulate primed pluripotency, including the fibroblast growth factor/extracellular signal-regulated kinase (FGF/ERK) and Activin/Nodal pathways 25 , 26 .

The establishment of naïve hESCs required the identification of culture conditions that could support the naïve state while suppressing the primed state. This requirement led researchers to focus on modulating signaling pathways, including the inhibition of the FGF/ERK 32 and transforming growth factor-beta (TGF-β)/Activin/Nodal pathways 33 , which are known for maintaining primed pluripotency. The application of small molecules to target these pathways enabled the conversion of primed hESCs into a state resembling naïve pluripotency 23 , 34 .

After years of extensive efforts, the landmark achievement of establishing naïve hESCs was made by Gafni et al. in 2013. Gafni and colleagues successfully established naïve hESCs using a combination of small molecules and growth factors 35 . They showed that inhibiting the FGF/ERK, TGF-β, and WNT/β-catenin pathways while simultaneously activating the LIF/STAT3 signaling pathway could prompt the conversion of primed hESCs to a naïve state 36 . These newly established naïve hESCs exhibited key features of naïve pluripotency, including the acquisition of epigenetic states similar to those of naïve mESCs, the expression of naïve-specific markers, and the attainment of an enhanced differentiation potential 23 , 35 .

Blastoids modeling early embryo development

The early stages of mammalian embryogenesis, leading up to implantation, encompass a series of conserved, systematic steps across various species, including ovulation, fertilization, cleavage, and implantation. Upon ovulation, the oocyte travels through the oviduct, where fertilization takes place in the ampulla. As the embryo travels toward the uterus, it undergoes asynchronous cleavages. At this juncture, the mammalian genome is activated, facilitating embryonic development through the expression of early transcribed proteins.

In humans, major waves of zygotic gene activation occur at the 8-cell stage, whereas in mice, activation arises at the 2-cell stage (minor waves transpire at the 4-cell stage and late zygotic stage for human and mouse embryos, respectively) 37 , 38 , 39 , 40 . Researchers have endeavored to model this process by generating human eight-cell-like cells (8CLCs) to probe the mechanism of totipotency 41 . In 2022, Mazid et al. developed a method to produce 8CLCs from human PSCs (hPSCs), revealing key roles of developmental pluripotency associated 3 (DPPA3), a master regulator of DNA methylation in oocytes, and tetrapeptide repeat homeobox 1 (TPRX1), a eutherian totipotent cell homeobox (ETCHbox) family transcription factor, in this process. Using this embryo model, the researchers demonstrated that 8CLCs can contribute to embryonic and extraembryonic lineages, providing a valuable resource for the study of the earliest stages of human embryogenesis.

Compaction is one of the most significant events of mammalian cleavage. In mice, after the embryo reaches the 8-cell stage, compaction occurs as blastomeres express cell adhesion proteins and coalesce into a sphere of cells 42 . This compact embryo matures into a 16-cell morula, where its inner cells later become constituents of the ICM, and its outer cells give rise to the trophectoderm (TE) 43 . The ICM is situated on one side within the TE, culminating in the formation of the blastocyst. In essence, the blastocyst comprises two primary cell populations: the ICM and the TE.

The peri-implantation period designates the phase during which the blastocyst is unattached in the uterus, preceding its implantation into the uterine wall, and differentiates into several fundamental structures. The nascent cells of the ICM segregate to form the primitive endoderm (PrE) and the epiblast. These intricate processes, which are fundamental to early embryonic development, have historically been difficult to study in depth. However, recent advances in stem cell research have now made it possible to create in vitro models that closely mimic these early stages of development (Fig. 2 ).

A depiction of the early developmental stages, spanning from the zygote phase (E0.5 of both mouse and human embryo) to the blastocyst stage (E5 and E7 of the mouse and human embryo, respectively), of mouse and human embryos are portrayed in the middle. In vitro models corresponding to the in vivo embryo of each developmental stage are illustrated on either side (top, mouse; bottom, human). Specific cells are color-coded throughout the figure (epiblast, green; hypoblast, pink; trophoblast, purple; ICM, yellow); the same color represents similar states at which they are in. Starting cells (C) of each model are noted adjacent to each illustration.

Since the isolation of mESCs in the 1980s 20 , 21 , stem cell research has significantly advanced. With the subsequent discovery of trophoblast stem cells (TSCs) 44 and extraembryonic endoderm stem cells (XENs) 45 , researchers have been equipped with essential cellular tools for the creation of in vitro mouse embryo-like structures known as mouse blastoids. This marked a pivotal breakthrough in embryonic research, signifying the first successful attempt to replicate an entire embryo in vitro.

Rivron et al. set a precedent by successfully generating the first mouse blastoids 46 . Following this breakthrough, numerous research groups have also developed similar in vitro structures that mimic in vivo blastocysts by employing a variety of methods 46 , 47 , 48 . Mouse blastoids are generated by aggregating various types of stem cells, including pluripotent and extraembryonic stem cells, in non-adherent hydrogel microwells, utilizing the self-organizing property of the mammalian embryo 43 . These cells are cultured with a blend of morphogens that influence the specification of the epiblast and TE lineages. Through this self-assembly process, in vitro structures that strikingly resemble in vivo blastocysts are created.

A pivotal event in successful blastoid formation is cavitation, underpinned by TE formation. TE formation is fostered by inhibiting the Hippo and TGF-β pathways. Concurrently, the addition of LIF and inhibitors of mitogen-activated protein kinase kinase (MEK) and glycogen synthase kinase 3 (GSK3) (known as 2i) helps maintain the pluripotency of ESCs by activating the STAT3, ERK, and WNT signaling pathways 49 . Moreover, Y-27632, a Rho-associated protein kinase (ROCK) inhibitor, is utilized to prevent cell apoptosis during this complex process.

In just 3 to 4 days, blastoids successfully take shape, exhibiting features such as the blastocoel cavity, ICM, and TE layer, the organization of which closely replicates the spatial organization seen in in vivo blastocysts. As cells start the process of self-assembly into blastoids, additional transcription factors, including GATA binding protein 6 (GATA6) and caudal type homeobox 2 (CDX2), are activated. These transcription factors govern lineage specification and cell differentiation, with GATA6 promoting the formation of the PrE lineage and CDX2 playing a crucial role in inducing the proper differentiation of the TE lineage 43 , 50 , 51 . The precise activation of transcription factors within blastoids is essential for directing the self-organization and lineage segregation of ESCs and TSCs into a fully functional blastoid structure. These blastoids share several characteristics with natural blastocysts, such as the capacity to acquire apical‒basal polarity and tight junctions.

Expanding upon strategies used in the creation of mouse blastoids, researchers have successfully generated human blastoids by harnessing the unique properties of hPSCs. Human naïve ESCs, notable for their increased plasticity and lower lineage barrier compared to naïve mESCs, have the capacity to differentiate into both embryonic (epiblast) and all extraembryonic (TE and hypoblast) lineages 52 , 53 , 54 . This significant capability allows the formation of human blastoids without necessitating the mixture of ESCs and TSCs 55 , 56 , 57 . In a parallel manner, human extended pluripotent stem cells (hEPSCs) 58 , 59 , cultured to embody developmental potency for embryonic and extraembryonic cell lineages, have shown the potential to generate blastoids 60 .

Several techniques have been developed to generate human blastoids. For instance, human blastoids can be generated by aggregating naïve hPSCs or hEPSCs in rounded microwells using centrifugation 55 , and similar results can be achieved with standard microwells or pyramid wells 56 , 57 , 60 . It should be noted that the use of a single cell type, such as naïve hPSCs or EPSCs, is not the only option to induce blastoid formation in the medium. Indeed, blastoids can also be generated through a combination of hEPSCs and TE-like cells derived from EPSCs 61 . Similarly, 8CLCs have been used to derive blastoids that exhibit both morphological and transcriptomic similarities to human blastocysts 41 .

Another breakthrough was the direct reprogramming of human somatic cells, which was achieved by infecting human dermal fibroblasts with a virus capable of inducing the expression of OCT4, SRY-box 2 (SOX2), Kruppel-like factor 4 (KLF4), and c-MYC, which in turn generated induced blastoids, or iBlastoids 62 . These human blastoids bear transcriptomic and morphological resemblances to human blastocysts, and remarkably, some have even shown the potential to initiate events akin to implantation.

Blastoids have the potential to be implanted into a hormone-stimulated endometrial layer 55 , allowing the observation of post-implantation processes. In compliance with the International Society for Stem Cell Research (ISSCR) guidelines 63 , which allow human experimental culture up to 14 days, these human blastoids could potentially be implanted into a synthetic endometrial lining for the purpose of investigating post-implantation events. The implantation of blastoids revealed that this process took place in the polar TE region, which likely became polarized due to its nascent relation to epiblast-like cells 55 . After implantation, sustained proliferation of epiblasts, TEs, and PrE-like cells was observed 55 .

Although several blastoids have cellular compositions similar to those of human in vivo blastocysts, it is necessary to investigate them more closely to determine whether they precisely replicate the in vivo state. The similarity of most of the blastoids created thus far to in vivo blastocysts has been validated using single-cell transcriptomics. Occasionally, however, the cells formed in blastoids do not match those of human early blastocysts but rather those of later developmental phases (such as gastrulation after embryonic day (E) 14) 64 . Therefore, further analysis is required to determine how accurately these in vitro models represent the in vivo context, and ongoing research should focus on increasing their similarity.

Early gastruloids recapitulating the formation of three germ layers

Gastrulation is one of the most important processes of the developing mammalian embryo, where it incorporates several combinations of cell movements in newly positioning the embryonic tissue to form the three germ layers—ectoderm, mesoderm, and endoderm—which set a platform that further enables the navigation of cellular lineage specification by placing the tissues accordingly throughout the anterior-posterior (AP), dorsal-ventral, and lateral (left and right) axes. By appropriately positioning the embryonic tissue layers, organizing axes, and facilitating progressive specialization, the embryo is able to undergo processes such as organogenesis and limb morphogenesis that lead to the formation of more complex structures.

Going back to mark the initiation of the creation of synthetic embryos, as mentioned earlier, developmental biologists Martin and Evans found that ES-cell-like 2D clonal pluripotent teratocarcinoma cells suspended into aggregates self-organize themselves to acquire a cellular organization pattern similar to that of the differentiation of the early mouse embryo 65 . Following their discovery, other studies have likewise continued to generate 3D aggregates that were able to recapitulate early embryonic differentiation and thus were collectively termed “embryoid bodies (EBs)”. When EBs derived from ES cells of mouse blastocyst ICM origin were cultured under in vivo-like conditions with appropriate developmental cues, they acquired the ability to differentiate into all three germ layers 66 , 67 .

Taking the potency of pluripotent stem cells to advantage, the addition of various signaling factors parallel to those that work at the corresponding developmental stage to EBs coaxed the formation of models that recapitulate the early development of the embryo. Due to the established role of signaling factors around this stage of embryonic development, small molecules added to induce the growth of in vitro models are short-range and similar among different protocols. Agonists of the FGF, BMP, and WNT signaling pathways (such as FGF2, BMP4, and CHIR99021, respectively)—key signaling pathways that govern development—as well as inhibitors (such as PD173074, LDN193189, and XAV939, respectively) are used to induce or inhibit characteristics of the developing embryo (Fig. 3 ).

In vivo developmental stages of the mouse and human embryos are depicted in the middle, from the process of gastrulation (E6.5 and E16 of the mouse and human embryo, respectively) to early organogenesis (E8.5 and E19 of the mouse and human embryo, respectively). In vitro models corresponding to the in vivo embryo of each developmental stage are illustrated on either side (top, mouse; bottom, human), as well as to note the original paper they were formed in. Specific tissues/organs are color-coded throughout the figure for both the embryos in gastrulation (mouse extraembryonic ectoderm, light purple; human extraembryonic ectoderm, dark purple; extraembryonic endoderm, pink; mesoderm, orange; endoderm, yellow; epiblast, blue) and those in early organogenesis (mouse extraembryonic ectoderm, light purple; extraembryonic endoderm, pink; brain, sky blue; spinal cord, turquoise; skin, dark navy; heart, red; notochord, dark red; somites, brown; presomitic mesoderm, dark orange; mesoderm, orange; gut and/or endoderm, yellow; neuromesodermal progenitors, green). Specific medium (M) or morphogens, including growth factors, cytokines, and inhibitors, used in the protocols for generating each in vitro model are listed adjacent to each gastruloid illustration.

As such, incorporating signaling pathways into in vitro cell aggregates yields models that recapitulate important hallmarks of gastrulation. For example, symmetry breaking is achievable in EBs in vitro, where the local activation of the WNT signaling pathway induces AP polarity and the formation of a primitive streak-like region, resulting in differentiation into a mesendodermal compartment (in contrast, neurectodermal differentiation is achieved by WNT signaling inhibition) 68 . Furthermore, the activation of WNT signaling at an appropriate time frame (at the initial stages of culture, which is usually within 74 h post-aggregation) induces the formation of a progressive elongating domain similar to that of the in vivo tail bud of the embryo, as well as the specification of the endoderm, which all lead to simulating aspects of the gastrulation period of the embryo 69 . Additionally, the precise timing regulated by WNT/β-catenin and Nodal signaling induces symmetry breaking in gastruloids even without the presence of extraembryonic tissue or localized signaling, allowing further polarization to take place, which is evidenced by distinct T/Brachyury expression 70 . Reducing FGF signaling yielded a diminished tail phenotype in the embryo models, indicating that the inducement of this signaling pathway is important for elongation 71 . With the knowledge of biological mechanisms underlying embryonic development and the use of key signaling factors, several models that mimic aspects of gastrulation were generated, starting from micropatterned 2D models (“2D gastruloids”), which encompass the endoderm, mesoderm, and ectoderm 72 , 73 , 74 , 75 , 76 , to more advanced free-floating 3D gastrulation models (“3D gastruloids”).

Early works to recapitulate aspects of gastrulation were conducted using mouse stem cells. The first absolute 3D gastruloid was created by van den Brink and colleagues in 2014 69 . Consisting of only 300 mouse ES cells, the EB was cultured in vitro to create a 3D gastruloid, where hallmarks of early mouse embryonic development, such as symmetry breaking and axial organization, were observable under the activation of WNT/β-catenin signaling, which further caused germ layer specification and minor axial elongation upon the addition of specific morphogens such as CHIR99021 and Activin A 77 . The usual 3D gastruloids are cultured through an initial few-hour-to-day EB suspension, whereafter they are then further matured under shaking culture conditions while adding certain morphogens to induce multi-axial organization and a temporal pattern similar to the gene expression of the embryo. Some gastruloids also acquire in vivo patterning integrity by expressing the collinear Hox gene pattern throughout the AP axis 77 .

Through the advancement of the creation of mESC embryonic models, adjustments in the culture conditions of these models enabled more developed structures as well as new discoveries. Although mouse gastruloids effectively mimic embryo-like axial morphogenesis and patterning, they lack regions such as the anterior embryonic region (which includes the brain). To improve this inaccurate depiction of the in vivo embryo, studies have inhibited WNT signaling during the early stages of development to induce gastruloids with anterior neural tissue 68 , 78 , since elevated expression of genes of the posterior mesoderm was observable with the inducement of WNT signaling 78 . However, WNT signaling is key in breaking axial symmetry in embryonic development, where its set dose induces primitive streak markers and spontaneously breaks AP symmetry. The aforementioned AP axis establishment enables the organization of embryonic tissues. Despite genetic studies that state that this process relies on the embryo’s exposure to extraembryonic spatiotemporally located signals—WNT/β-catenin and Nodal signaling—studies performed with mouse gastruloid models proved that this might not necessarily be the case. Mouse ESC-based gastruloids induced without surrounding extraembryonic tissues showed localized T/Brachyury expression with polarity and extension to one side, suggesting the notion of AP axis development. Thus, this discovery suggested that extraembryonic tissues are not necessarily required for the embryo to undergo self-patterning 69 .

Gastrulation occurs at approximately 16 days post-fertilization (dpf) in human embryos, and in vitro biomimetic models that recapitulate this stage are also achievable by utilizing primed hESCs and hiPSCs, enabling the formation of human gastruloids without the violation of major ethical issues. As in the case of initial murine gastruloid models, human gastruloids were also developed under 2D conditions that portrayed aspects of gastrulation, such as the development of primitive streak-like structures and the three germ layer domains 72 , 79 .

The development of 3D human gastruloids facilitated the study of human development, as they more accurately depict the in vivo gastrulation process than earlier 2D models. Human PSCs embedded in the matrix, such as by utilizing the commercial extracellular matrix (ECM) formula Matrigel, and cultured under conditions that are supplemented with the right morphogens construct aggregates that undergo symmetry breaking and gene expression patterning 80 . For example, the addition of BMP and WNT agonists was found to be crucial in the establishment of AP polarity and the elongation of the tail bud 81 . Adding extraembryonic compartments resulted in models that more closely resembled the in vivo gastrulating embryo 82 by mimicking attachment and AP symmetry breaking and further exhibiting human gastrula-specific cells. 3D aggregates that broke symmetry formed extensions along the AP axis with organized germ layers 83 , 84 . Through the development of the technology of generating human gastruloids, specific gastruloids that addressed parts of the early stages of post-implantation human embryonic development were created, such as the processes of primitive streak formation 80 , neurulation 84 , 85 , 86 , and somitogenesis 87 .

Late gastruloids modeling somitogenesis and organogenesis

At E7.5 in mice and approximately 20 dpf in humans, the presomitic mesoderm (PSM) condenses and gives rise to somites along the lateral sides of the central neural tube in an anterior-to-posterior fashion. These somites later form structures that act as anchors that hold parts of the embryo, such as the vertebrae, skeletal muscles, cartilage, and dermis. Shortly after somitogenesis has begun, organogenesis is launched, with the heart being the first organ to be generated. Models that mimic somitogenesis, as well as organogenesis, have been created over recent years (specific models are further discussed in the section “Modeling congenital diseases using synthetic embryos”). Segmentation clock waves are observable in mesoderm-based hPSC models (termed “axioloids” or “somatoids”) that mimic segmentation and somitogenesis in the human embryo 88 . Simpler models that recreate oscillatory movements were also generated 89 . Inducing AP symmetry breaking in hPSC models allowed scientists to study the mechanisms underlying human somitogenesis.

Gastruloids have also been able to acquire regions that resemble the gut tube. The formation of the AP and dorsal-ventral axes enables the patterning of the primitive gut tube in mouse gastruloids 90 . More specifically, the anterior foregut, midgut, and hindgut were induced in the gastruloid through the creation of a primordium that covers the overall region of the structure 91 . Furthermore, several gastruloid models, such as trunk-like structure (TLS) gastruloids (using mESCs) 92 and elongating multi-lineage organized (EMLO) gastruloids (using hiPSCs) 84 , portray gut tube-like structures.

Development of synthetic whole embryos

Instead of models that recapitulate only a small aspect of the early developing embryo, attempts were made to model its overall characteristics. In mice, the cylindrical shape of the post-implantation embryo is due to the polar TE invagination and actomyosin contractility that give rise to the extraembryonic ectoderm 39 . Stem cell models that resemble these early “egg-cylinders” were generated by combining mESCs with TSCs and incorporating tissues such as those that resemble the extraembryonic ectoderm without recapitulating the blastocyst stage 93 . Adding XEN cells that resemble extraembryonic endoderm cells to these models (ETX or iETX embryos) enabled the production of a visceral endoderm-like epithelium that lines the in vitro conceptus 94 , 95 . When synthetic embryos were constituents of extraembryonic compartments, they could initiate implantation 95 and further induce gastrulation, neurulation, and organogenesis 96 .

Several culture techniques that assist in establishing proper embryonic development ex vivo were created, such as circulator systems 97 and roller culture systems 98 . By taking advantage of these systems, the process of generating whole embryos in vitro was conducted. Due to the aforementioned ISSCR regulation restricting the culture of human embryos to a maximum of 14 days in vitro for research purposes (the “14-day rule”), attempts to create synthetic whole embryos in long-term culture conditions were generally proceeded using mESCs.

To create synthetic whole embryo models, an environment similar to in vivo conditions is needed. With the adaptation of the electronically controlled ex utero roller device that previously cultured a mouse embryo until E11 by the incorporation of the ex utero culture medium (EUCM), which is a mixture of 50% rat serum, 25% Dulbecco’s modified Eagle’s medium (DMEM), and 25% human umbilical cord blood serum 99 , mouse naïve ESCs were cultured until E8.5, which yielded a mouse synthetic whole embryo model that was able to undergo gastrulation and to form organ precursors 100 .

Attempts to create human synthetic whole embryos were also made. For human synthetic whole embryo models, blastoids are currently the most advanced tool, mimicking the entire pre-implantation stage of the human embryo. Although researchers have made attempts to push these blastoids toward the post-implantation stages in vitro, they have not been able to match them with the development of in vivo blastocysts 55 , 56 , 60 , 61 , 62 . Recently, there have been strides toward creating a human synthetic whole embryo that can model more advanced developmental processes mimicking post-implantation stages while maintaining the morphological integrity of a natural embryo.

Two groups of scientists have recently reported their attempts at creating human post-implantation embryo models 101 , 102 . They drew on strategies previously used to create a mouse synthetic whole embryo, which involved differentiating embryonic and extraembryonic cells separately before aggregating them to produce a post-implantation embryo. Weatherbee et al. generated two types of extraembryonic-like cells from hESCs through the overexpression of transcription factors: GATA6 and SRY-box transcription factor 17 (SOX17) for the hypoblast and GATA3 and transcription factor AP-2 gamma (TFAP2C) for TSCs 101 . On the other hand, Oldak et al. utilized ectopic expression of lineage-promoting transgenes to induce the formation of the hypoblast and trophoblast 102 .

Both groups reported human synthetic whole embryo models that replicated the hallmarks of 13-14 dpf human embryos. Weatherbee et al.’s model consisted of structures such as the amnion, extraembryonic mesenchyme, and primordial germ cell-like cells 101 . Oldak et al. reported the formation of the bilaminar disc, epiblast lumenogenesis, amniogenesis, primordial germ cell specification, yolk sac formation, and the expansion of extraembryonic mesoderm in their model 102 . While the creation of human synthetic whole embryo models is expected to continue to evolve, it is essential to closely monitor these developments within the framework of ethical considerations and international agreements.

Modeling congenital diseases using synthetic embryos

One of the main advantages of synthetic embryos is their feasibility for modeling diseases that arise during the early stages of embryonic development. Thus, congenital diseases that occur due to initial abnormal development are of interest. Furthermore, because synthetic embryo models such as gastruloids can be easily manipulated to imitate specific stages of development, diseases that occur at a particular event could be observed. Although only a few- considering the subject’s relatively recent advancement into the field, attempts have been made to incorporate synthetic embryoids to model human diseases. This section includes an overview of models that recapitulate key processes of embryonic development with respect to disease modeling (Fig. 4 ).

Cardiovascular defects (left), diseases associated with somitogenesis disruption (middle), and neurulation defects (right) may be modeled by current gastruloids. Appropriate gastrulation models are depicted in each category.

Congenital heart disease (CHD) is one of the leading causes of death in newborns 103 . Several models that mimic the heart have been made to recapitulate aspects of cardiogenesis 104 , 105 , 106 , 107 , 108 ; however, to fully understand the dynamics of heart development, observing the organ alone is insufficient. Taking advantage of the gastruloid characteristic of encapsulating multiple cell types of all germ layers, cardiogenesis development is more accurately portrayed by gastruloid models since the formation of the heart necessitates complex interactions such as those among the organ, cardiac progenitor cells, and endothelial cells. Recent studies have mimicked cardiogenesis by manipulating embryo models by incorporating well-known cardiogenic growth factors, such as vascular endothelial growth factor 165 (VEGF-165), FGF2, and ascorbic acid. Rossi et al. captured the process of cardiogenesis via mESC gastruloids, in which the spatiotemporal accuracy was highly conserved compared to its in vivo counterpart 109 . The model incorporates both cardiac progenitors of the first heart field (FHF) and the second heart field (SHF), which anteriorly organize into a cardiac crescent-like region and form tissues that imitate the beating of the heart.

Expanding upon synthetic embryo models that recapitulate only cardiogenesis, new models that include both cardiac muscle and neurons to portray neuro-cardiac lineages have been made. Olmsted and Paluh have added the aspect of cardiogenesis 110 to the “elongating multi-lineage organized” (EMLO) gastruloids that they had previously generated 111 , creating EMLOC gastruloids that enable the remodeling of both cardiogenesis and neurogenesis to explain the complex interconnected lineages between the processes. Various developmental features that resemble those of the in vivo heart were observed, including the formation of the heart tube, putative outflow tract, ventricular wall, and epicardium, as well as the differentiation of cardiomyocytes. This neuro-cardiac model may offer a platform to provide further understanding of cardiac diseases such as neural-based heart arrhythmia diseases and congenital heart diseases.

Vertebrates acquire segmented structures through a process called somitogenesis. This process occurs in early development when somites, blocks of paraxial mesoderm, form on either side of the neural tube and the notochord in a bilateral position along the AP axis. Presenting phenotypes similar to those of mouse embryos, mouse gastruloids have been offered as appropriate models to study vertebrate segmentation. For instance, genetic alteration of gastruloids yielded phenotypes that matched those of previous knockout mouse embryo models. Veenvliet et al. used mESCs deleted with the T-box transcription factor 6 ( Tbx6 ) gene to generate gastruloids (TLSs) 92 . The resulting phenotype was the loss of somites and ectopic neural tubes, which correlates with the in vivo phenomenon of PSM transdifferentiation and ectopic neural tube formation at the expense of somites and PSM. Van den Brink et al. performed a screening test that showed that decreased levels of FGF signaling resulted in the generation of fewer somites, which was similar to the defects observed in Fgf mutant mice, and that incorporating 10% Matrigel may induce somitogenesis in gastruloids 71 .

Aberrations in somitogenesis can lead to several diseases, such as vertebral malformations and spinal defects. Specifically, Yamanaka et al. created axioloids that recapitulated human somitogenesis in vitro and applied them to investigate congenital diseases associated with the human spine 88 . They introduced loss-of-function mutations in the segmentation clock gene hes family bHLH transcription factor 7 (HES7) and the marker for the anterior PSM mesoderm posterior bHLH transcription factor 2 (MESP2), which are associated with segmentation defects of the vertebrae (SDV), and generated axioloids with them. Two different HES7 knockout hiPSC lines were used to create axioloids, and their phenotypes were assessed. There were losses of segments and rostro-caudal patterning, as well as the formation of the epithelial somites. Regarding the oscillatory activity of the segmentation clock, Yamanaka and colleagues found a definite loss of HES7 oscillation. In axioloids derived from iPS cell lines introduced with a point mutation in HES7, there were also losses of rostro-caudal patterning, yet these models managed to express normal HES7 oscillation in the tail bud as well as a few somitic mesoderm markers. MESP2 is known to be mutated in patients with SDV. Yamanaka and colleagues also assessed the effects of axioloids with MESP2 knockout. MESP2-knockout axioloids also lacked segments and epithelial somites and exhibited abnormal rostro-caudal patterning, despite their normal elongation. However, MESP2 knockout did not result in the loss of oscillation of HES7. As such, these axioloids serve as models that offer insights into the segmentation development of the human body and a platform to study the pathogenesis of the spine.

Neurulation is a highly orchestrated and complex morphogenetic process in which the neural plate fuses to form the neural tube, the precursor of the brain and spinal cord of the central nervous system. When the process of neurulation is disrupted, severe congenital abnormalities such as neural tube defects (NTDs) arise. Several studies have shown that gastruloids are capable of recapitulating the formation of the neural tube. Libby et al. generated a human neural tube gastruloid that demonstrated aspects of early spinal cord development, such as axial elongation, neuromesodermal progenitor (NMP) cell population maintenance, and neuroepithelial cell generation 81 . With exposure to doses of the WNT agonist CHIR99021, the elongation of hPSC aggregates and the formation of NMP cells could be observed, as well as the presence of the mesoderm and neuroectoderm. Neural tubes were also induced in mouse TLSs created by Veenvliet et al. when cultured in a 5% growth factor-reduced Matrigel condition, as well as by the addition of the WNT agonist CHIR99021 and the BMP signaling inhibitor LDN193189 92 .

Furthermore, neurulation was depicted in partially 3D gastruloids induced by 2D micropatterning by Karzbrun et al., where a neural-tube-like structure was detectable 85 . In this model, Karzbrun and colleagues incorporated three small-molecule inhibitors—the ROCK inhibitor Y-27632, the HSP-90 inhibitor novobiocin, and the NTD-associated drug valproic acid—to test the suitability of these gastruloids for modeling NTDs. The application of the small molecules resulted in morphological abnormalities, including less curvature of the tube and overall folding defects, which indicated that their model may indeed be utilized to represent the neural tube to study defects to some extent.

Perspectives

Historically, investigating human development has been an ongoing endeavor with significant limitations, including restricted understanding due to the complexity of the process, the lack of appropriate models (not to mention the inaccessibility of human embryos), and ethical concerns. Herein, we have illustrated how the generation of synthetic models of early mammalian development has assisted in overcoming these challenges.

Through the discovery of stem cells and research progress made due to their manipulation, the scope of human embryo experimentation, once available only through direct practice on authentic embryos, became more diverse. By inducing human stem cells to become structures characterized by organized systems, the need for human embryos has diminished, thereby compensating for the inaccessibility of human embryos due to the shortage of samples and ethical issues. Ethical concerns are inevitable; accordingly, regulations have been made by officials such as the ISSCR with recurring discussions among scientists 112 .

Synthetic models do not act merely as models that recapitulate early development; they also provide a platform for which numerous diseases, and their underlying mechanisms, could be further studied. Since these early embryo models may reliably replicate the embryo’s state at certain developmental stages, specific diseases that arise at specific time points may be investigated by recreating a developmentally similar embryonic structure with temporal accuracy. For example, blastoids allow the further study of implantation mechanisms, which then may suggest possible factors associated with implantation failures, and gastruloids may model congenital diseases, including but not limited to those stated above. Current mammalian embryo models are able to recapitulate in vivo embryos of Carnegie stages 1 to 10, in which diseases that arise may be modeled.

As a shared advantage of in vitro structures, synthetic embryo models may be produced in bulk, offering compatibility with systems that require large amounts of samples, such as drug screening and toxicological assays. By using stem cell-based models, researchers have studied the effects of small molecules on mammalian development 113 , 114 . Furthermore, teratogenicity could be tested through quantifiable gastruloids, which yield statistical robustness, and further applications may be tested, such as species-specific reactions to the molecules 115 .

Nonetheless, a limitation that remains is the total inducement of the synthetic whole embryo in vitro. Several attempts have been made to create synthetic whole embryos, yet results show that there is still a need for further understanding of developmental mechanisms, given the morphological difference of the cultured in vitro embryo with little resemblance to the in vivo embryo and the fact that these synthetic embryos cannot further develop after a set time—not to mention their incompetence to be born.

Furthermore, it should be noted that many in vitro early embryogenesis models representing various developmental stages and dimensions often lack fidelity to in vivo conditions in their cellular composition. Recent work 64 has revealed that several in vitro models are composed of cells that differ significantly from the transcriptome of their in vivo counterparts. For modes of fidelity validation, the current tools we have to validate the integrity of cell lineages in in vitro models are largely limited to single-cell analyses. At present, single-cell transcriptomics represents the most advanced tool to assess the fidelity of these models. However, even if parts of the in vitro model display gene expression patterns similar to those of its in vivo counterpart, reliance on single-cell transcriptomics alone is insufficient. The number of genes considered per cell in a single-cell analysis ranges from as few as 200 genes to an average of 2000–6000 genes, suggesting that comparisons of similarities between in vitro models and their in vivo counterparts cannot be completely accurate considering the approximate 30,000 genes in the human genome. Much like how the invention of telescopes unveiled previously unseen galaxies and microscopes provided insights into cellular structures, the advancement of tools that govern degrees of accuracy for analyzing differences between in vitro and in vivo embryos will enable us to see new perspectives that lie within the two objects. Thus, there is a clear need for a more sophisticated tool capable of accurately discerning even the subtlest differences. Such innovation would enable the creation of in vitro models that more closely—if not exactly—mirror the in vivo embryo. Further technological improvements would enhance investigations in modeling diseases and pragmatic applications of these models to clinical areas, such as cell-type-specific transplantation in regenerative medicine.

Given that previous works were conducted on less relevant structures via 2D culture and that the field of stem cell and developmental biology has made substantial progress since these advancements in pursuit of generating proper mammalian in vitro embryos that accurately recapitulate developmental stages elicit anticipations of further research improvements and discoveries. The availability of once-seemingly impossible technical applications of lineage-specific stem cell differentiation via these advances will enable further research into discovering new mechanisms that underlie the development of the embryo.

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Acknowledgements

This research was supported by grants (NRF-2022R1A2C300270212, Bio&Medical Technology Development Program: RS-2023-00223277) from the National Research Foundation of Korea funded by the Korean government (MSIT), Samsung Science and Technology Foundation (SSTF-BA2101-12), New Faculty Startup Fund from Seoul National University, the BK21FOUR Research Fellowship, and Samsung Advanced Institute of Technology (IO220818-02090-01).

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Yunhee Kim, Inha Kim & Kunyoo Shin

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Kim, Y., Kim, I. & Shin, K. A new era of stem cell and developmental biology: from blastoids to synthetic embryos and beyond. Exp Mol Med 55 , 2127–2137 (2023). https://doi.org/10.1038/s12276-023-01097-8

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