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Stem cell biology and regenerative medicine are the interdisciplinary biological and clinical sciences that study how self-renewing cells of the body can be made, maintained, directed, and used to repair or replace damaged tissue. For much of the twentieth century, the prevailing view in developmental biology was that cellular differentiation was effectively irreversible, so that a cell that committed to a lineage, such as a skin cell or a neuron, kept that identity for life. This view was challenged by nuclear transfer experiments, by the isolation of embryonic stem cells, and by the discovery of induced pluripotent stem cells (iPSCs). These findings established that differentiation is a reversible process of epigenetic and transcriptional regulation, and they opened the field of regenerative medicine.

Somatic Cell Nuclear Transfer and Pluripotent Stem Cell Isolation

The idea that the nucleus of a differentiated cell retains the full genome was tested by Robert Briggs and Thomas King in the 1950s and was shown by John Gurdon in 1962, who transplanted nuclei from tadpole intestinal cells into enucleated eggs of the frog Xenopus laevis and obtained normal tadpoles. The cloning of the sheep Dolly in 1996 by Ian Wilmut and colleagues, by somatic cell nuclear transfer from an adult mammary cell, showed that the same is true in mammals. Mouse embryonic stem cells were first isolated in 1981 by Martin Evans and Matthew Kaufman and, independently, by Gail R. Martin. James Thomson derived human embryonic stem cells in 1998. In 2006, Shinya Yamanaka and Kazutoshi Takahashi reported the reprogramming of mouse fibroblasts to pluripotency, and human iPSCs followed in 2007 from the groups of Yamanaka and Thomson. Gurdon and Yamanaka shared the 2012 Nobel Prize in Physiology or Medicine for the discovery that mature cells can be reprogrammed to become pluripotent.

A stem cell is defined by two properties:

1. Self-renewal: The ability to divide to produce at least one daughter cell that retains the stem cell state, so that the stem cell population is maintained over long periods. This can occur by symmetric division, which produces two stem cells, or by asymmetric division, which produces one stem cell and one more differentiated cell.
2. Potency: The ability to differentiate into specialized cell types. Potency is classified by the range of types that a cell can produce.

Pluripotency is the ability to give rise to all of the cell types of the adult body, which derive from the three germ layers. In the embryo, pluripotent cells exist only transiently. The functional tests of pluripotency are the formation of teratomas that contain tissues of all three layers when cells are injected into immunodeficient mice, the contribution to all tissues of a chimeric embryo, and, for mouse cells, the generation of an entire animal by tetraploid complementation. The last test cannot be applied to human cells for ethical reasons, so human pluripotency is judged by teratoma formation, differentiation assays, and molecular criteria.

States of Pluripotency

Pluripotent cells exist in more than one state. Mouse embryonic stem cells derived from the inner cell mass of a blastocyst are in a "naive" state that corresponds to the pre-implantation epiblast, and mouse cells from the post-implantation epiblast (epiblast stem cells) are in a "primed" state. Conventional human embryonic stem cells and iPSCs are closer to the primed state. Culture conditions that support a naive-like state in human cells have been developed, and they are used to study early development and X-chromosome regulation. The core pluripotency network includes the transcription factors OCT4 (encoded by POU5F1), SOX2, and NANOG, which activate pluripotency genes and repress genes for differentiation.

Sources of Pluripotent Cells

Embryonic stem cells (ESCs) are derived from the inner cell mass of blastocyst-stage embryos, which are usually surplus embryos from in vitro fertilization. They have a high capacity for self-renewal and differentiation, and they have great therapeutic potential. Their use raises ethical questions, because derivation requires the destruction of an embryo, and the views and the regulations differ widely among countries. The cells are also genetically different from the patient, so transplanted cells can be rejected by the immune system unless immunosuppression is used or the cells are matched or modified.

Reprogramming

Takahashi and Yamanaka screened 24 candidate genes for the ability to induce pluripotency in mouse fibroblasts, and they found that four transcription factors were sufficient: Oct4, Sox2, Klf4, and c-Myc (OSKM, the Yamanaka factors). When these are expressed in a differentiated somatic cell, the cell is reprogrammed to a state similar to that of an embryonic stem cell, with similar gene expression, and the ability to differentiate into all three germ layers. The resulting cells are called induced pluripotent stem cells. The process is slow and inefficient, and only a small fraction of cells (often below 1 percent) become iPSCs over a few weeks. Reprogramming occurs in stages that include a loss of the somatic gene program, a mesenchymal-to-epithelial transition, activation of the pluripotency network, and global changes in chromatin and DNA methylation. The process is described as the erasing of much of the epigenetic memory of the original cell, but the erasure is not always complete. iPSCs can retain residual DNA methylation and gene expression patterns of the cell type of origin, which can bias their differentiation toward related lineages.

Alternatives to the original factor set have been developed. The Thomson group used OCT4, SOX2, NANOG, and LIN28. MYC is an oncogene, and it can be omitted, which lowers efficiency and the risk of tumors. Small molecules, microRNAs, and chemical-only methods have also induced pluripotency, and chemical reprogramming of human cells was reported in 2022.

Methods of Delivery

The first iPSCs were made with retroviral vectors that integrate into the genome, which raises the risk of insertional mutagenesis and of reactivation of the transgenes. Methods that do not integrate are now preferred for clinical research. They include Sendai virus (an RNA virus), episomal plasmids, synthetic modified mRNA, and protein delivery. The source cells are commonly skin fibroblasts, blood cells such as T cells and peripheral blood mononuclear cells, and cells from urine.

Direct Reprogramming

Cells can also be converted from one differentiated type to another, without passing through a pluripotent state, which is called direct reprogramming or transdifferentiation. Examples are the conversion of fibroblasts to cardiomyocyte-like cells, to neurons, or to hepatocyte-like cells by sets of lineage-specific transcription factors. In vivo reprogramming of cells in an organ has been studied as a way to regenerate tissue without transplantation, but it is at an early stage.

Advantages of iPSCs

• Autologous therapy. iPSCs can be made from a patient's own cells, differentiated into the needed cell type, and transplanted back. In principle the cells match the patient's genome, which reduces the risk of immune rejection. In practice, autologous production is slow and expensive (many months and a high cost for each patient), and some studies have found that iPSC-derived cells can still provoke immune responses, because of epigenetic changes or abnormal expression of some proteins. For this reason, many programs make "off-the-shelf" allogeneic lines of cells from donors that are selected to match common human leukocyte antigen (HLA) types, or edited to avoid immune recognition.
• Disease modeling. Cells from a patient with a genetic disease, such as amyotrophic lateral sclerosis, Alzheimer's disease, or long QT syndrome, can be reprogrammed and differentiated into the affected cell type, for example neurons or cardiomyocytes, to model the disease in a dish. This permits the study of mechanisms in human cells and the testing of drugs. The patient's genome can be edited with CRISPR-Cas9, and isogenic lines that differ only in the mutation are used as controls. Models in the dish are limited, because the cells are often immature and resemble fetal cells more than adult ones, and they lack the tissue context and the effects of ageing.
• Drug screening and toxicity testing. iPSC-derived cells, such as cardiomyocytes and hepatocytes, are used to test the effects and the toxicity of compounds.
• Organoids. Pluripotent stem cells can form three-dimensional organoids, which resemble parts of organs such as the brain, intestine, kidney, and retina, and they are used to study development and disease.

Risks and Challenges

• Tumorigenicity. Undifferentiated pluripotent cells that remain in a graft can form teratomas, so the purity of differentiated products must be tested. Reprogramming factors such as MYC and the culture of the cells can also cause genetic and epigenetic abnormalities.
• Genomic instability. Cultured pluripotent cells can acquire chromosomal abnormalities and mutations, including mutations in TP53, and this requires monitoring of the genome of the cell lines.
• Variability. Different iPSC lines differ in their ability to differentiate, because of their origin, genetic background, and culture conditions.
• Maturity. Differentiated cells often have an immature, fetal-like phenotype.
• Manufacturing and regulation. Clinical use requires reproducible production under good manufacturing practice, with quality control and safety testing.

Hematopoietic, Epidermal, and Limbal Stem Cell Transplants

Not all stem cell therapies use pluripotent cells. Hematopoietic stem cell transplantation, which uses stem cells from bone marrow, mobilized peripheral blood, or umbilical cord blood, has been in clinical use since the 1950s and 1960s and is the best-established stem cell treatment, for example in leukemia and some inherited blood disorders. Epidermal stem cells cultured from a small skin biopsy are used to produce sheets of skin for severe burns, and limbal stem cells are used to restore the cornea in some patients. Mesenchymal stromal cells have been tested in many conditions, but the evidence of benefit is limited for most. Many clinics worldwide offer unproven stem cell treatments. The International Society for Stem Cell Research and regulators warn that they can cause harm.

The Triad of Cells, Biomaterial Scaffolds, and Signals

Regenerative medicine aims to restore normal function by replacing or regenerating human cells, tissues, or organs. Tissue engineering is the application of this aim that combines cells, materials, and biochemical and physical signals. Three components are usually needed:

1. Cells. These are often stem cell-derived cells, such as iPSC-derived cardiomyocytes for repair of the heart after a myocardial infarction, or primary cells from the patient, such as chondrocytes for cartilage.
2. Scaffold. Cells in the body attach to the extracellular matrix (ECM), which provides structural support and biochemical and mechanical cues. Scaffolds are made from biocompatible and often biodegradable polymers or natural materials such as collagen, fibrin, and hyaluronic acid, or from decellularized tissue, in which the cells of a donor organ are removed and the ECM is retained. The stiffness, porosity, and architecture of the scaffold influence cell behavior, including differentiation.
3. Signals. Growth factors and mechanical stimulation, such as the cyclic stretching of growing muscle or the flow of fluid, are applied in bioreactors to promote maturation, alignment, and function as a coherent tissue.

3D Bioprinting and the Microvascular Diffusion Limit

Additional approaches include three-dimensional bioprinting, which deposits cells and materials (bioinks) in a defined pattern, cell sheets that are stacked without a scaffold, and the growth of organs in animals from human cells in chimeras, which is an experimental approach that raises ethical questions.

A central difficulty is the supply of nutrients and oxygen. Tissues thicker than about 100 to 200 μm need a vascular network, because diffusion cannot supply the cells at greater distances from a capillary. The engineering of large, vascularized, whole organs, such as liver or kidney, is still far from clinical use, and no engineered solid organ of this size is in routine use. Simpler or thinner tissues are already used clinically, such as skin substitutes and cultured epidermal grafts, cartilage repair products, engineered cornea, and some blood vessels and tracheal and bladder constructs.

Retinal, Neurological, and Endocrine Progenitor Trials

Clinical trials of cells derived from pluripotent stem cells have begun. Early trials have treated retinal diseases such as macular degeneration with retinal pigment epithelium cells derived from embryonic stem cells or iPSCs, Parkinson's disease with dopamine neuron precursors, type 1 diabetes with pancreatic islet cells or precursors, spinal cord injury, and heart failure. A first trial in Japan transplanted iPSC-derived retinal cells in 2014, and a trial of dopaminergic progenitors for Parkinson's disease has also been reported. The results so far are early evidence on safety and in some cases of function. Large trials that show benefit are needed, and the status of individual trials changes quickly, so current sources should be checked.

How do the four Yamanaka factors (Oct4, Sox2, Klf4, c-Myc) erase somatic cell identity?

During iPSC reprogramming, Oct4, Sox2, and Klf4 act as pioneer transcription factors that bind nucleosomal DNA at closed chromatin sites, recruiting histone remodelers and TET DNA demethylases to silence somatic lineage enhancers and activate the endogenous pluripotency network (POU5F1, SOX2, NANOG), while c-Myc accelerates cell cycle progression and global chromatin accessibility.

What is the mechanistic difference between naive and primed pluripotency?

Naive pluripotent stem cells correspond to the pre-implantation blastocyst inner cell mass, exhibiting global DNA hypomethylation, two active X chromosomes in female cells (X_aX_a), and dependence on LIF/STAT3 signaling. Primed pluripotent cells correspond to the post-implantation epiblast, exhibiting higher DNA methylation, X-chromosome inactivation (X_aX_i), and dependence on FGF2 and Activin/Nodal signaling.

Why can engineered tissues thicker than 200 micrometers not survive without blood vessels?

According to Fick's laws of diffusion, dissolved oxygen and glucose diffusing from surrounding interstitial fluid or culture medium are consumed by outer cell layers faster than they can penetrate deeper than 100 to 200 μm. Beyond this diffusion limit, core cells experience severe hypoxia and metabolic necrosis unless perfused by a hierarchical capillary microvascular network.

• Stem cell
• Induced pluripotent stem cell
• Embryonic stem cell
• Regenerative medicine
• Tissue engineering
• Cellular differentiation
• Organoid