In this section, we discuss two types of pluripotent mammalian cells: embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. Our focus is on the network of genes and proteins that regulate the pluripotent state of these cells and can subsequently lead to multiple types of differentiated cells. In culture, these two types of pluripotent cells can be used to form specific types of differentiated cells for research purposes or, potentially, as “replacement parts” for worn out or diseased cells in patients. iPS cells can be formed from patients with many types of diseases and then differentiated into the specific cell type affected by the disease; here we see how study of such cells can illuminate crucial underlying causes of a specific individual’s disease.
The Inner Cell Mass Is the Source of ES Cells
Embryonic stem cells can be isolated from the inner cell mass of early mammalian embryos and grown indefinitely in culture when attached to a feeder-cell layer that provides certain essential growth factors (Figure 1a). As mentioned in the chapter introduction, cultured ES cells are pluripotent: they can differentiate into a wide range of cell types of the three primary germ layers, either in culture or after reinsertion into a host embryo. More specifically, mouse ES cells can be injected into the blastocoel of an early mouse embryo and the cell aggregate surgically transplanted into the uterus of a pseudopregnant female. The injected ES cells will paticipate in forming most, if not all, tissues of the resultant chimeric mice. Furthermore, the injected ES cells will often give rise to functional sperm and eggs that, in turn, can generate normal live mice.
EXPERIMENTAL Fig1. Embryonic stem (ES) cells can be maintained in culture and can form differentiated cell types. (a) Human or mouse blastocysts are grown from cleavage-stage embryos produced by in vitro fertilization. The ICM is separated from the surrounding extraembryonic tissues and plated onto a layer of fibroblast cells, which help to nourish the embryonic cells by providing specific protein hormones. When individual cells are replated, they form colonies of ES cells, which can be maintained for many generations and can be stored frozen. ES cells can also be cultured without a fibroblast feeder layer if specific cytokines are added; leukemia inhibitory fac tor (LIF), for instance, supports growth of mouse ES cells by triggering activation of the Stat3 transcription factor; see J. S. Odorico et al., 2001, Stem Cells 19:193. (b) Embryonic stem cells allowed to differentiate in suspension culture become multicellular aggregates termed embryoid bodies. (c) Hematoxylin- and eosin-stained sections of embryoid bodies that contain derivatives of all three germ layers that are formed from the ICM during embryogenesis. Arrows in the images point to the following tissue types: (left) gut epithelium (endoderm), (middle) cartilage (mesoderm), and (right) neuroepithelial rosettes (ectoderm). Black bar = 100 μm. [Parts (b) and (c) courtesy of Dr. Lauren Surface and Dr. Laurie Boyer.]
In a more recent variation on these experiments, the host blastocyst is treated with drugs that transiently block mitosis so that its cells become tetraploid (with four copies of each chromosome, incapable of forming differentiated cells and tissues), in contrast to the diploid ES cells that are injected into the blastocyst. In this case, all the cells in the live mice that are born after transplantation of the blastocyst aggregate derive from the donor ES cells. This finding is powerful evidence that single mouse ES cells are indeed pluripotent. Because ethical considerations and, in many countries, legal restrictions preclude similar transplantation experiments with human ES cells, formal proof that they are pluripotent is lacking.
Importantly, both human and mouse ES cells can differentiate into a wide range of cell types in culture. When cultured in suspension, ES cells form multicellular aggregates, called embryoid bodies (Figure 1b), that resemble early embryos in the variety of tissues they form. When embryoid bodies are subsequently treated with various combinations of growth factors or transferred to a solid surface, they produce a variety of differentiated cell types, including gut epithelia, cartilage, and neural cells (Figure 1c). Under other conditions, ES cells have been induced to differentiate in culture into precursors for various specific cell types, including blood cells and pigmented epithelia; for this reason, ES cells have proved extremely useful in identifying the factors that commit a pluripotent cell to differentiating down a particular cell lineage.
What properties give these cells of the early embryo their remarkable plasticity? As we’ll see in the next section, a variety of actors play a role: DNA methylation, transcription factors, chromatin regulators, and micro-RNAs all affect which genes become active.
Multiple Factors Control the Pluripotency of ES Cells
During the earliest stages of embryogenesis, as the zygote be gins to divide, both the paternal and maternal DNA become demethylated. This happens in part because a key maintenance methyl transferase, Dnmt1, is transiently excluded from the nucleus and in part because demethylase enzymes actively re move or “erase” methylation marks from 5-methyl cytosine residues during early development. As a result, the pattern of DNA methylation is reset during the first few cell divisions, erasing earlier epigenetic marking of the DNA and creating conditions in which cells have greater potential for diverse pathways of development. Mice engineered to lack Dnmt1 die as early embryos with drastically undermethylated DNA. ES cells prepared from such embryos are able to divide in culture, but in contrast to normal ES cells, cannot undergo in vitro differentiation.
ES cell properties are also critically dependent on the action of master transcription factors produced shortly after fertilization. The transcription factors Oct4, Sox2, and Nanog have essential roles in early development and are re quired for the specification of ICM cells in the embryo as well as for the specification of ES cells in culture. The expression of Oct4 and Nanog is exclusive to pluripotent cells such as the cells of the ICM and cultured ES cells. Sox2 is found in pluripotent cells, but its expression is also necessary in the multipotent neural stem cells that give rise exclusively to neuronal and glial cell types. Genetic studies in the mouse suggest that these three regulators have distinct roles, but may function in related path ways to maintain the developmental potential of pluripotent cells. For example, disruption of Oct4 or Sox2 results in the inappropriate differentiation of ICM and ES cells into trophectoderm. However, forced expression of Oct4 in ES cells leads to a phenotype that is similar to that caused by loss of Nanog function. Thus knowledge of the set of genes regulated by these transcription factors might reveal their essential roles during development.
The genes that are bound by these three transcription factors have been identified using chromatin immunoprecipitation experiments; each protein is found at more than a thousand chromosomal locations. The target genes encode a wide variety of proteins, including the Oct4, Nanog, and Sox2 proteins themselves, forming an autoregulatory loop in which each of these three transcription fac tors induces its own expression as well as that of the others (Figure 2). These transcription factors also bind to the transcription-control regions of many genes encoding proteins and micro-RNAs important for the proliferation and self-renewal of ES cells.
Fig2. Transcriptional network regulating pluripotency of ES cells. Each of three master transcription factors, Oct4, Sox2, and Nanog, binds to its own promoter as well as to the promoters of the other two (black lines), forming a positive autoregulatory loop that activates transcription of each of these genes. These transcription factors also bind to the transcription-control regions of many active genes encoding proteins and micro-RNAs important for the proliferation and self-renewal of ES cells as well as to those of many genes that are silenced in undifferentiated ES cells and that encode proteins and micro-RNAs essential for the formation of many differentiated cell types (magenta lines). See L. A. Boyer et al., 2006, Curr. Opin. Genet. Devel. 16:455–462.
Several protein hormones are provided by feeder cells or added to culture media to prevent differentiation of ES cells. These hormones include leukemia inhibitory factor (LIF), which activates Stat3; Wnt, which activates the β-catenin transcription factor; and bone morphogenetic protein 4 (BMP4), which activates the Smad1 transcription factor (see Chapter 16). In ES cells, these three transcription factors bind at multiple genomic sites co-occupied by Oct4, Nanog, and Sox2 proteins. Thus signaling pathways activated by cell-surface receptors are directly coupled to regulation of genes in the core pluripotency circuitry; this observation reinforces a point made in Chapter 16 that transcription fac tors activated by cell-surface receptors frequently bind at sites in the genome occupied by master transcription factors specific to that type of cell.
Chromatin regulators that control gene transcription are also important in ES cells. In Drosophila, Polycomb group proteins form complexes to maintain gene repression states that have been previously established by DNA-binding transcription factors. Two mammalian protein complexes related to the fly Polycomb proteins, PRC1 and PRC2, are abundant in ES cells. Early mouse embryos lacking components of PRC2 display early developmental defects. The PRC2 complex acts by adding methyl groups to lysine 27 of histone H3, thus altering chromatin structure to repress genes. (Note that the methylation here is on an amino acid in a protein, a type of regulation distinct from the methylation of cytosine residues in DNA.) In ES cells, PRC1 and PRC2 both silence genes whose encoded proteins or micro-RNAs (miRNAs) would otherwise induce differentiation into particular types of cells; the Poly comb proteins also maintain these genes in an epigenetic “preactivation” state such that they are poised to become activated later as part of the proper execution of specific developmental gene expression programs. Thus ES cells lacking PRC2 functions fail to differentiate properly.
Many other regulators play important roles in control ling gene expression and maintaining pluripotency during very early development. For example, the gene encoding the miRNA let-7 is transcribed in ES cells, but the precursor RNA transcript is not cleaved to form the mature, functional miRNA. ES cells express a developmentally regulated RNA-binding protein termed Lin28 that binds to the let-7 precursor RNA and prevents its cleavage. Experimental expression of mature let-7 miRNA in ES cells blocks their ability to undergo self-renewal, and thus repression of let-7 processing by Lin28 is essential for pluripotency.
As we will see later, the possibility of using embryonic stem cells therapeutically to restore or replace damaged tissue is fueling much research on how to induce them to differentiate into specific cell types. Apart from their possible benefit in treating disease, ES cells have already proved invaluable for producing mouse mutants useful in studying a wide range of diseases, developmental mechanisms, behavior, and physiology. Using the recombinant DNA techniques described in Chapter 6, one can eliminate or modify the function of a specific gene in ES cells. The mutated ES cells can then be employed to produce mice with a gene knock out. Analysis of the effects of deleting or modifying a gene in this way often provides clues about the normal function of the gene and its encoded protein.
Animal Cloning Shows That Differentiation Can Be Reversed
Although different cell types may transcribe different parts of the genome, for the most part the genome is identical in all cells. Segments of the genome are rearranged and lost during development of the T and B lymphocytes of the immune system from hematopoietic precursors, but most somatic cells appear to have an intact genome, equivalent to that in the germ line. Evidence that at least some somatic cells have a complete and functional genome comes from the successful production of cloned animals by nuclear transfer. In this procedure, often called somatic-cell nuclear transfer (SCNT), the nucleus of an adult somatic cell is introduced into an egg whose nucleus has been removed; the manipulated egg, which contains the diploid number of chromosomes and is equivalent to a zygote, is then implanted into a foster mother. The only source of genetic information to guide development of the embryo is the nuclear genome of the donor somatic cell. The low efficiency of generating cloned animals by SCNT, combined with a high frequency of diseases such as obesity in the animals that are cloned, however, raises questions about how many adult somatic cells do in fact have a complete functional genome and whether those that do can be completely reprogrammed into a pluripotent undifferentiated state. Even the successes, such as the famous cloned sheep “Dolly,” have some medical problems. Even if differentiated cells have a physically complete genome, clearly only parts of it are transcriptionally active. A cell could, for example, have an intact genome, but be unable to properly reactivate specific genes due to inherited chromatin epigenetic states.
Further evidence that the genome of a differentiated cell can revert to having the full developmental potential characteristic of an ES cell comes from experiments in which olfactory sensory neurons—postmitotic cells that normally will not divide again—were genetically marked with green fluorescence protein (GFP) and then used as donors of nuclei (Figure 3). When the nuclei from differentiated olfactory sensory neurons were implanted into enucleated mouse oocytes, a small fraction of them developed into blastocysts that produced GFP. The blastocysts were used to derive ES cell lines, which were then used to generate mouse embryos. These embryos, derived entirely from olfactory sensory neuron genomes, formed healthy green-fluorescing mice. Thus, at least in some cases, the genome of a differentiated cell can be reprogrammed completely to form all tissues of a mouse.
EXPERIMENTAL Fig3. Mice can be cloned by ( d ) somatic-cell nuclear transfer from olfactory neurons. (a) Procedure for generating cloned ES cell lines using nuclei from olfactory sensory neurons and using them to generate cloned mice. Step 1: A nucleus from an olfactory sensory neuron isolated from a mouse that expressed green fluorescent protein (GFP) only in its olfactory neurons was used to replace the nucleus of a mouse egg, and the resultant zygote was cultured to the blastocyst stage (step 2). The ICM cells, all of which were clones of the original olfactory sensory neuron, and all of which expressed GFP, were used to generate lines of ES cells (step 3). Step 4: These ES cells were injected into a tetraploid blastocyst. Step 5: When the blastocyst was transplanted into the uterus of a pseudopregnant mouse, the tetraploid cells from the host blastocyst could form the placenta (gray), but not the embryo proper; therefore, all of cells in the embryo proper and in the mouse that developed from it expressed GFP (step 6). (b–c) Bright-field (top) and fluorescence images (bottom) of (b) nuclear-transfer blastocysts and (c) the ES cells that were isolated from the ICM. (d) A control 12-hour-old mouse (top) and a mouse cloned from an olfactory sensory neuron, all of whose cells expressed GFP (bottom). [Parts (b–d) reprinted by permission from Macmillan Publishers Lt, from Eggan, K., et al., “Mice cloned from olfactory sensory neurons,” Nature, 2004, 428(6978):44–9.]
Somatic Cells Can Generate iPS Cells
Because of the inefficiency of somatic-cell nuclear trans fer, it remained unclear whether all types of somatic mammalian cells retained an intact genome and whether they could be induced to dedifferentiate into an ES cell–like state. Shinya Yamanaka used retrovirus vectors to express a wide variety of transcription factors, singly and in combination, in cultured fibroblast cells. Remarkably, he found that both human and mouse fibroblasts could be reprogrammed to a pluripotent state, called an induced pluripotent stem-cell state, similar to that of an embryonic stem cell, by transformation with retroviruses encoding just four proteins: KLF4, Sox2, Oct4, and Myc. Note that two of these, Sox2 and Oct4, are two of the master transcription factors expressed in ES cells, as discussed previously. In addition to fibro blasts, keratinocytes (skin-forming cells) and other types of differentiated cells have been reprogrammed to iPS cells. Like ES cells, single mouse iPS cells can be experimentally introduced into a blastocyst and form all of the tissues of a mouse, including germ cells, attesting to the fact that somatic cells can indeed be reprogrammed to an embryonic pluripotent state.
Several other transcription factors, and even certain small organic molecules, can replace the Oct4 gene in the Yamanaka reprogramming “cocktail.” Subsequent analysis led to the discovery that each of these factors directly activates transcription of the endogenous (cellular) Oct4 gene, leading to induction of pluripotency. Thus it was hypothesized that, over time, forced expression of transcription factor genes activates expression of many cellular genes, including those encoding Oct4 and other pluripotency proteins; over the course of several weeks, this activation reprograms the somatic cells to an ES-like state. To experimentally establish the point that activation of endogenous genes leads to reprogramming to an ES-like state, cultured keratinocytes were repeatedly transfected with synthetic mRNAs encoding the four canonical Yamanaka transcription factors, KLF4, Sox2, Oct4, and Myc. These cultured cells generated normal iPS cells that had no trace of any of the exogenously added mRNAs, attesting to the reprogram ming of keratinocytes into iPS cells by inducing expression of only normal cellular genes.
In fibroblasts, the chromatin of most pluripotency- associated genes is inaccessible to transcription-factor binding, primarily due to the repressive histone H3 lysine 9 trimethylation (H3K9Me3) mark. Among the genes activated by Oct4 are two that encode H3K9 demethylases, which remove these repressive chromatin marks and, over time, result in activation of pluripotency genes. Consistent with this notion, expression of these H3K9 demethylases increases during reprogramming, and their knockdown inhibits efficient iPS-cell generation. Indeed, reprogramming involves major changes in epigenetic modifications, including DNA methylation and several other types of histone modifications that serve to repress or allow potential activation of hundreds of genes.
Because iPS cells can be derived from somatic cells of patients with difficult-to-understand diseases, they have already proved invaluable in uncovering the molecular and cellular basis of several afflictions (Figure 4). Consider amyotrophic lateral sclerosis (ALS), often called Lou Gehrig’s disease, a fatal disease in which the motor neurons that connect the spinal cord to the muscles of the body progressively die off, causing muscle weakness and death, limb paralysis, and ultimately death due to respiratory failure. There is no cure.
Fig4. Medical applications of iPS cells. In this example, the patient has a neurodegenerative disorder caused by abnormalities in certain nerve cells (neurons). Patient-specific iPS cells—in this case derived by recombinant expression of transcription factors in cells isolated from a skin biopsy—can be used in one of two ways. In cases in which the disease-causing mutation is known (for example, familial Parkinson’s disease), gene targeting could be used to repair the DNA sequence (right). The gene-corrected patient-specific iPS cells would then undergo directed differentiation into the affected neuronal subtype (for example, midbrain dopaminergic neurons) and be transplanted into the patient’s brain (to engraft the nigrostriatal axis). Alternatively, directed differentiation of the patient-specific iPS cells into the affected neuronal subtype (left) will allow the patient’s disease to be modeled in vitro, and potential drugs can be screened, aiding in the discovery of novel therapeutic compounds. See D. A. Robinton and G. Q. Daley, 2012, Nature 481:295.
In approximately 10 percent of patients, the disease is dominantly inherited (familial ALS), but in 90 percent of patients, there is no apparent genetic linkage (sporadic ALS). An analysis of the underlying causes of the disease at a molecular and cellular level was impossible for many years because one cannot simply extract neurons or the surrounding glial cells from living humans and analyze or culture them.
In about 20 percent of patients with familial ALS, there is a point mutation in the gene SOD1, encoding Cu/Zn superoxide dismutase 1; the mutant SOD1 protein forms aggregates that can damage cells. About 40 percent of patients with familial ALS and 10 percent of patients with the noninherited form have a mutation in the C9ORF72 gene (of unknown function; called chromosome 9 open reading frame 72). This mutation also often occurs in people with frontotemporal dementia, the second most common form of dementia after Alzheimer's disease, explaining why some people develop both diseases simultaneously. The mRNA transcribed from normal human C9ORF72 genes has up to 30 repeats of the hexanucleotide GGGGCC, but mutant ALS-causing genes can have up to thousands of these repeats.
In several studies, iPS cells derived from the skin cells of elderly patients with these and other familial and sporadic forms of the disease were successfully differentiated in culture to form motor neurons; this success demonstrated the feasibility of leveraging the self-renewal of iPS cells to generate a potentially limitless supply of the cells specifically affected by ALS. One study showed that motor neurons bearing several types of ALS mutations were hyperexcitable, generating more of the electrical signals called action potentials than normal. This excess excitability also caused the neurons to make more errors in protein folding and accumulate misfolded proteins, leading to aberrant cell function. In iPS-derived neurons from patients with the C9ORF72 mutation, the RNAs containing the large numbers of repeating GGGGCC sequences were in aggregates, bound to multiple RNA-binding proteins important for normal cell functions; this binding prevented these proteins from catalyzing key steps in the production of other cellular mRNAs. Overall, the C9ORF72 mutation made the motor neurons produce abnormal amounts of many other cellular RNAs and made the cells very sensitive to stress.
In a separate study to dissect the molecular cause of ALS, motor neurons were generated from human ES or iPS cells and cultured with primary human astrocytes, a type of glial cell that surrounds neurons and regulates several of their functions. Many of the motor neurons died if the astrocytes expressed the mutant form of SOD1, but not if they expressed the wild-type form, suggesting that at least in this familial form of ALS, the defective cells are both astrocytes and motor neurons. Indeed, astrocytes ex pressing the mutant form of SOD1 secreted protein factors that were toxic to adjacent motor neurons.
In these and several other studies, researchers screened thousands of small organic molecules, including many ap proved as drugs for treatment of other unrelated diseases, for those that could reverse the abnormalities in the ALS iPS cell–derived motor neurons. Several were identified and are in clinical trials to see if they can slow or stop the devastating effects of ALS. In any case, these experiments illustrate the value of iPS and ES cells in generating cell culture models of many types of difficult-to-study human diseases that can be used to screen for drugs that could treat many as yet un treatable afflictions.
ES and iPS Cells Can Generate Functional Differentiated Human Cells
Neurons and glial cells, as well as other cell types, derived from human iPS cells have been implanted into mice with some promising results. Stem cell–derived cardiomyocytes (heart muscle cells) can correct heart arrhythmias; certain glial cells—oligodendrocytes—show promise in aiding recovery from experimental spinal injury; and retinal epithelial cells can partially correct defects in mouse models of blindness.
One very recent advance—the generation of normal insulin-secreting β islet cells from human iPS and ES cells— shows promise for treatment of both type 1 and type 2 diabetes. Type 1 diabetes results from autoimmune destruction of pancreatic β cells, whereas the more common type 2 diabetes results from insulin resistance in liver and muscle, eventually leading to dysfunction and death of β cells. Patients who receive transplants of human islets from cadavers can be made insulin independent for 5 years or longer, but this approach is limited because of the scarcity and quality of donor islets; thus the possibility of an unlimited supply of human β cells from stem cells could potentially extend this therapy to millions of new patients.
One key to this successful generation of β cells was employing successive treatment with different combinations of growth factors that stimulated iPS or ES cells to traverse the normal embryonic developmental sequence by which the progeny of undifferentiated ICM cells form β cells (Figure 5a). The so-called SC-β cells that resulted have a structure very similar to that of normal β islet cells, including secretory granules filled with almost crystal line insulin; they also secrete normal amounts of insulin in response to elevation of the glucose level in their culture medium. Shortly after their transplantation into mice, these cells secrete human insulin into the serum in a glucose-regulated manner. Most important, after transplantation of these cells into immunocompromised diabetic mice, their high glucose levels are lowered to nor mal (Figure 5b), indicating the potential use of these islet cells—which can be produced in culture in essentially un limited numbers—for the treatment of diabetes. Screening to identify new drugs that improve β cell function, survival, or proliferation can also make use of such a uniform supply of stem cell–derived β cells.
Fig5. Production of normal insulin-secreting β islet cells from human iPS or ES cells. (a) Schematic of directed differentiation of human ES or iPS cells into insulin-secreting β islet cells. Clusters of a few hundred human ES or iPS cells were sequentially cultured in media containing the indicated growth factors for the indicated number of days to first produce definitive endoderm cells, then a series of pancreatic progenitor cells, then pancreatic endocrine progenitors, and finally stem cell–derived insulin-producing β islet cells (termed SC-β cells). Act A, activin A; CHIR, GSK3 inhibitor; KGF, keratinocyte growth factor; RA, retinoic acid; SANT1, Sonic Hedgehog pathway antagonist; LDN, a BMP type 1 receptor inhibitor; PdbU, a protein kinase C activator; Alk5i, Alk5 receptor inhibitor II; T3, triiodo thyronine, a thyroid hormone; XXI, γ-secretase inhibitor; betacellulin, an EGF family member. (b) SC-β cells can be used to treat diabetes in mice. These experiments used a strain of diabetic mice with a muta tion in the insulin gene as well as mutations in several immune-system genes such that the animals did not reject transplants of human tissue. Previous work had shown that the elevated glucose levels in these mice could be restored to normal by transplantation with human pancreatic islets. In this experiment, mice were transplanted with SC-β cells (black circles) or a similar number of control pancreatic progenitor cells (white circles). At the start of the experiment, the average blood glucose level in these mice was about 11 mM, well above the normal 5 mM. The average blood glucose level in the control mice rose continuously to about 30 mM, indicating severe diabetes, while in the mice transplanted with the human SC-β cells, blood glucose dropped to nearly the normal 5 mM. [Part (b) data from F. Pagliuca et al., 2014, Cell 159:428.]
These SC-β cells are assuredly a harbinger of what is to come. The coming years are certain to see the development of many other types of differentiated cells from human iPS cells that can be used as “replacement parts” for a variety of maladies. Many important questions must be answered, however, before the feasibility of using human ES or iPS cells for therapeutic purposes can be assessed adequately. For instance, when undifferentiated human or mouse ES or iPS cells are transplanted into an experimental mouse, they form teratomas, tumors that contains masses of partially differentiated cell types. Thus it is essential to ensure that all of the ES or iPS cells used to generate an implant have indeed undergone differentiation and have lost their pluripotency and their ability to induce teratomas or cause other problems.
