Extrinsic regulation of pluripotent stem cells Martin F. Pera 1 & Patrick P. L. Tam 2,3

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1 NATURE Vol June 2010 doi: /nature09228 REVIEW INSIGHT Extrinsic regulation of pluripotent stem s Martin F. Pera 1 & Patrick P. L. Tam 2,3 During early mammalian development, as the pluripotent s that give rise to all of the tissues of the body proliferate and expand in number, they pass through transition states marked by a stepwise restriction in developmental potential and by changes in the expression of key regulatory genes. Recent findings show that cultured stem- lines derived from different stages of mouse development can mimic these transition states. They further reveal that there is a high degree of heterogeneity and plasticity in pluripotent populations in vitro and that these properties are modulated by extrinsic signalling. Understanding the extrinsic control of plasticity will guide efforts to use human pluripotent stem s in research and therapy. Pluripotent stem s have two remarkable properties: immortality, or the capacity for indefinite self-renewal; and pluripotency, the ability to give rise to all the tissues of the adult body. The derivation of pluripotent stem s from the human embryo 1,2 (human embryonic stem (ES) s) and the development of pluripotent stem s (induced pluripotent stem (ips) s) through the reprogramming 3 of adult human s 4,5 are seminal technological breakthroughs that hold the promise of revolutionizing biomedical research (see page 704). Studies of the molecular basis, be it genetic or epigenetic, of these natural and induced pluripotent states, as well as investigations into how pluripotency is maintained and the mechanisms of lineage commitment, are important not only for improving the understanding of mammalian embryogenesis and ular differentiation but also for developing successful stem--based therapies for regenerative medicine. Pluripotency is a transitory state of embryonic s that exists only during a brief window of development. Shortly after the onset of embryogenesis, the s of the embryo, which are totipotent, become restricted in their developmental potential, becoming either progenitors that form extra-embryonic tissues (the placenta and fetal extra-embryonic membranes) or pluripotent progenitors, which form the three primary germ layers, from which all of the tissues of the fetus are formed. By contrast, stem s derived from these embryonic s can be maintained indefinitely in the pluripotent state in vitro. However, in mice, in which all of the stages of embryogenesis are experimentally accessible, different types of stem, with distinct phenotypes, have been derived from the embryo, suggesting that these s have reached different states of developmental potential and that the pluripotent state is a continuum of states. Although it seems that the different types of embryo-derived stem have a common genetic network of transcription factors that maintains them in a pluripotent state, recent studies have shown that these s have a highly plastic phenotype. Certain stem- types readily convert into other stem- types, and many types of stem and their descendants can interconvert in response to extraular signals. The response to extrinsic signalling is crucial for understanding plasticity in pluripotent stem- populations, because extrinsic signals can be propagated through intraular signal-transduction pathways that converge on the genetic network that controls pluripotency. In vivo, stem- plasticity might enable the body to compensate for ular loss or developmental delay in early embryogenesis, as well as to respond to the vastly changing demands for production during tissue maintenance, remodelling, regeneration and repair in adults. In this Review, we discuss the multiple states of pluripotency in stem s, as well as the signalling systems that maintain these states. We focus on human pluripotent stem- regulation and draw on relevant findings from studies of mouse stem s. Recent surprising findings about how stem s are regulated in both species are providing insight into the multiple states of pluripotency. Pluripotent stem s in mice and humans Over the past few years, studies by several groups have provided a clear definition of the molecular phenotype of human ES s and have examined the relationship between human ES s and mouse pluripotent stem s in vitro and in the early embryo. Molecular phenotype of human ES s Human ES s, like other stem- populations, are characterized by their developmental potential, transcriptional and epigenetic profiles, and -surface markers. Developmental potential is assessed by a biological assay of the ability of the to give rise to all of the types in the body. This is, arguably, the definitive measure of pluripotency, but it is also the most difficult to measure in the case of human s. For mammalian stem s, two tests have traditionally been used to assess pluripotency. When injected into ectopic sites in host animals, pluripotent s form benign growths called teratomas, which contain multiple types of differentiated tissue representative of all three embryonic germ layers. Human ES s can also be tested in this way. The definitive test for pluripotency, which is applicable only in animal models, is the generation of germline-competent chimaeras after the introduction of stem s into pre-implantation embryos that are subsequently allowed to develop to full term in foster mothers. The contribution of the stem s to the chimaera not only reveals the extent to which s can differentiate to form all of the body s tissues but also provides proof of the functional capacity of the descendants of the stem s. It remains untested whether pluripotent stem s from non-human primates can participate in chimaera formation. The characterization of s on the basis of their immunological or molecular features is more straightforward, and such studies have yielded a high-resolution annotation of the phenotype of human ES s. More importantly, examination of the whole transcriptome, proteome and epigenome of human ES s has provided insight into 1 Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine, University of Southern California, Los Angeles, California 90033, USA. 2 Embryology Unit, Children s Medical Research Institute, Westmead, New South Wales 2145, Australia. 3 Discipline of Medicine, Sydney Medical School, University of Sydney, Sydney, New South Wales 2006, Australia. 713

2 INSIGHT REVIEW NATURE Vol June 2010 Table 1 Properties of various pluripotent populations grown in vitro Type of stem Stem- genes Cell-surface markers Response to factors Developmental potential Oct4 Nanog Sox2 Klf4 Dppa3 Rex1 Gbx2 Fgf5 SSEA1 SSEA3, SSEA4 Alkaline phosphatase LIF Nodal and/or activin FGF2 Teratoma Chimaera Mouse ES s X X X* X Mouse EPL s ND X X X X ND ND X Mouse FAB-SCs ND ND ND ND X ND X X Mouse EpiSCs X X X X X X X X Human ES s X X X ND Tick ( ) means that the gene or -surface marker is expressed, the indicated growth factors are required for self-renewal, or the indicated type will form teratomas or participate in chimaera formation. Cross (X) means that the gene or -surface marker is not expressed, the indicated factors are not required for self-renewal, or the indicated type will not form teratomas or participate in chimaera formation. ND (not determined) means that the attribute was not examined. EpiSC, epiblast stem ; EPL, early primitive ectoderm-like ; FAB-SC, stem obtained by culturing blastocysts in medium containing fibroblast growth factor 2 (FGF2), activin and the glycogen synthase kinase 3β (GSK3β) inhibitor BIO; SSEA, stage-specific embryonic antigen. *One study shows long-term self-renewal of mouse ES s in activin. Cells grown in leukaemia inhibitory factor (LIF) revert to an ES--like state. Cells are derived and maintained in these factors, but dependence on the factors for self-renewal has not been rigorously examined. The requirement for FGF2 has not been rigorously determined for these s. how pluripotency is regulated at the molecular level through common genetic networks 6,7. Whereas many of the early studies of human ES phenotype and gene expression were descriptive, recent molecular and genetic studies of pluripotent stem s in vitro, and their counterparts in the peri-implantation (around the time of implantation) embryo in mammals in vivo 8,9, are leading to a better understanding of the mechanisms of pluripotency maintenance and lineage commitment in mouse and human ES s. The human ES- transcriptome and epigenome have now been characterized: multiple data sets from microarray studies of human ES s have recently been subjected to a meta-analysis 10. Furthermore, a study of a large panel of stem- lines for their expression of genes that maintain pluripotency and control early lineage commitment has identified a subset of these genes whose expression pattern was highly correlated among lines of different origins 11. This finding suggests that expression of these genes robustly defines a human ES. A diverse and expanding set of -surface markers is used to charac terize human ES s. In general, these markers are defined by their ability to bind to monoclonal antibodies specific for glyco lipids or transmembrane glycoproteins of unknown function. A panel of these markers is used to define stem- lines derived from human blastocysts, as well as ips s, and distinguishes these s from the pluripotent stem- types derived from mice (Table 1). A core set of canonical markers is expressed with a high level of consistency across the range of available human ES- lines, which were independently derived using a variety of techniques 11 and are representative of diverse genetic backgrounds. Importantly, some of these markers are expressed in the inner mass of human blastocysts, suggesting that human ES s have a similar surface phenotype to the pre-implantation epiblast, in the blastocyst 12. Mouse pluripotent stem s How does the phenotype of human ES s relate to that of mouse pluripotent stem- populations? There is a core set of transcription factors that functions to maintain the pluripotent state in all of these populations. OCT4 (also known as POU5F1), NANOG and SOX2 co-occupy promoter regions of genes that are involved in pluripotency maintenance and early lineage differentiation, maintaining the pluripotency genes in an active state and repressing the lineage-specific genes. This set of pluripotency-associated transcription factors is expressed in all pluripotent stem- types that have been studied so far in humans and mice. These types are distinguished by their expression of other genes, however (Table 1). Many of the genes that are characteristic of human ES s, such as OCT4 (ref. 13) and DNMT3B (which encodes a DNA methyltransferase) 14, have been detected in the inner mass of human blastocysts, similarly to the -surface markers discussed above 15. These data are limited, however, and further study of human and non-human primate embryos in the pre- and post-implantation period are required to determine the developmental stage to which human ES s most clearly correspond. The mouse counterparts of these pluripotency transcription factor genes are also expressed in the pluripotent s in the mouse embryo. So far, three types of pluripotent and two types of lineage-restricted stem can be derived from early mouse embryos directly or through interconversion in vitro (Fig. 1). The pluripotent types are ES s, early primitive ectoderm-like (EPL) s 16 and epiblast stem s (EpiSCs) 17,18. Another type, FAB-SCs, is nullipotent (unable to differentiate) but can be converted to pluripotency in vitro. The lineage-restricted stem types are trophoblast stem (TS) s and extra-embryonic endoderm (XEN) stem s, which give rise to trophectoderm and to primitive endoderm and its derivatives, respectively. Under culture conditions that block key signalling pathways involved in lineage specification, ES s can be derived at a high frequency from the pre-implantation epiblast of the mouse blastocyst 19. EPL s 16 are generated from ES s in vitro under certain conditions, and their patterns of gene expression (Table 1) resemble the epiblast of the early post-implantation embryo (the early epiblast). Unlike ES s, they cannot participate in chimaera formation after injection into host blastocysts. EPL s might be viewed as an intermediate between ES s and EpiSCs, but a more direct comparison of these two types is warranted. Mouse EpiSCs are derived directly from the early epiblast, and their properties differ markedly from those of ES s. EpiSCs can also be derived from the late epiblast (in the gastrulating embryo) 20. FAB-SCs can be derived from the blastocyst through culture in the presence of fibroblast growth factor 2 (FGF2), activin and the non specific inhibitor of glycogen synthase kinase 3β (GSK3β) called BIO. These s have similar gene expression patterns to those of ES s (Table 1). They are nullipotent but can attain the pluripotent state through conversion to an ES--like intermediate 21. The properties of these mouse stem- types are thought to reflect their embryonic of origin in vivo, in addition to any adaptive changes made in response to propagation in vitro. From recent data, there is a strong case that mouse ES s are the in vitro equivalent of the epiblast of the pre-implantation embryo 22. This stage of the epiblast is derived from the inner mass and appears after the formation of the first two extra-embryonic lineages, the trophectoderm and the primitive endoderm. With some exceptions, the gene expression patterns and biological properties of mouse EpiSCs are broadly similar to those expected of the early post-implantation epiblast 17,18. Cells of the late epiblast (of the gastru lating embryo), from which EpiSCs can also be generated 20, undergo lineage commitment driven by FGFs, WNTs and bone morphogenetic proteins (BMPs) and their antagonists, which are all produced by the extra-embryonic tissues (the extra-embryonic ectoderm and visceral endoderm) (Fig. 1) and the epiblast itself. These recent findings on the developmental status of different types of mouse pluripotent stem have allowed comparative analysis of mouse stem s and human ES s. Human ES s and their mouse counterparts It has been argued that human ES s more closely resemble EpiSCs than the other pluripotent types derived from the mouse embryo. Indeed, some aspects of the growth requirements of human ES s 714

3 NATURE Vol June 2010 REVIEW INSIGHT Blastocyst Pre-gastrulation embryo Early gastrulation embryo Epiblast Extra-embryonic ectoderm Extra-embryonic ectoderm Primitive endoderm Trophectoderm Implantation Visceral endoderm Early epiblast Gastrulation Primitive streak Late epiblast ES s EPL s FAB-SCs TS s XEN s TS s EpiSCs EpiSCs Figure 1 Stem- types derived from mouse embryos around the time of implantation. At implantation, mouse blastocysts comprise three distinct types: the trophectoderm; the inner mass, which produces the primitive endoderm; and the naive (or early pre-implantation) epiblast. Under appropriate in vitro culture conditions, three types of stem with a comparable potential for differentiation to each of the types of the blastocyst can be derived: ES s, trophoblast stem (TS) s and extraembryonic endoderm (XEN) stem s 1 5,66, Another type of stem, FAB-SCs, can be obtained by culturing blastocysts in medium containing FGF2, activin and the GSK3β inhibitor BIO 25. And a further stem- type, EPL s, can be derived from ES s by culturing them in a -lineconditioned medium 21. TS s and XEN stem s are committed to form extra-embryonic tissues only. After implantation, the blastocyst grows into a pre-gastrulation embryo, which comprises the extra-embryonic ectoderm and the early epiblast, with the visceral endoderm enveloping both tissues. Pluripotent stem s can be derived from the epiblast of the early post-implantation embryo at days post coitum 22,23. Like ES s, these epiblast stem s (EpiSCs) are pluripotent in that they differentiate into the full range of typical germ-layer tissues in vitro and into teratomas in vivo. Epiblast s are progressively restricted in differentiation potential as they are allocated to the mesoderm and endoderm through ular ingression at the primitive streak during gastrulation. Despite the onset of tissue commitment at early gastrulation, EpiSCs can still be derived from the late epiblast of the embryo, the s of which have been experimentally shown by lineage analysis and fate-mapping studies to retain plasticity in fate. and EpiSCs in vitro (discussed in the next section) are similar and distinguish them from mouse ES s. For example, both human ES s and mouse EpiSCs 17,18 require nodal or activin signals to maintain their pluripotent state, whereas mouse ES s do not. Neither is maintained in the pluripotent state by leukaemia inhibitory factor (LIF), which is required for maintaining mouse ES s. Both grow poorly after dissociation into single s, and both respond similarly to culture conditions that drive the differentiation of germ-layer derivatives and extra-embryonic tissues, again in contrast to mouse ES s 23. The similarity between EpiSCs and human ES s makes for a compelling argument that they are counterparts, but there are crucial differences between the -surface marker and gene expression of the two types (Table 1). Mouse EpiSCs express the glycolipid epitope stage-specific embryonic antigen 1 (SSEA1) on their surface and lack alkaline phosphatase. By contrast, human ES s do not express SSEA1 but express alkaline phosphatase. Furthermore, in common with mouse ES s, human ES s express DPPA3 (also known as STELLA) 24 and KLF4 (ref. 25), whereas mouse EpiSCs express neither gene. This last difference is particularly important because these two genes have pivotal roles in the conversion between mouse ES s and EpiSCs. Dppa3 is activated on interconversion of mouse EpiSCs to ES s 26, and Klf4 overexpression can drive mouse EpiSCs to acquire ES- properties 27. In addition, human ES s express the gene encoding the transcription factor REX1 (also known as ZFP42) 11,28, which is present in mouse ES s but not EpiSCs, and they do not express the gene encoding the EpiSC marker FGF5 (refs 10, 11). In addition to stem- gene and -surface marker expression, a close analysis of the growth requirements of the two types also places some caveats on the conclusion that human ES s and mouse EpiSCs are developmentally equivalent. There is no strong evidence that mouse EpiSCs have a strict requirement for FGF2 for maintenance, whereas human ES s do 29. Moreover, responding to activin, which both of these types do, is not a definitive criterion for the pluripotent state. In this regard, one study has shown that activin could maintain ES s derived from the mouse inner mass in serum-free culture 30, although signalling involving the transcription factors SMAD2 and/or SMAD3 (the pathway used by nodal and activin) might not usually be required for mouse ES- maintenance 31. It has been suggested that mouse EpiSCs and human ES s have a similar developmental potential to that of the epiblast of mouse embryos at the post-implantation pre-gastrulation stage and gastrulation stage: namely, the ability to form tera tomas but an inability to contribute to the germ line after introduction into the blastocyst. It is notable, however, that both types can give rise to both trophectoderm and extraembryonic endoderm in vitro, a finding that is not easily reconciled with the idea that the s represent an in vitro equivalent of the late mouse epiblast, which has lost the ability to form these lineages in vivo. In addition, EpiSCs express transcription factors that are characteristic of early lineage commitment 18. Human ES s also express such factors, but this feature may be a characteristic of the heterogeneity of types in culture (see the section Plasticity of embryo-derived stem s in vitro ). Given the lack of data on gene expression during crucial phases of immediate post-implantation development in humans, it remains unclear precisely which embryonic is the counterpart of the human ES. In cases in which morphological data (mostly at the level of light and electron microscopy) allow human and mouse development to be compared, it is apparent that, despite broad similarities, there are important differences between these species in the timing of developmental milestones, the overall geometry of the embryo, and the emergence and morphology of the extra-embryonic tissues 32,33. Finally, there is a high degree of heterogeneity in human ES- cultures (see the section Plasticity of embryo-derived stem s in vitro ), suggesting that these populations represent a developmental continuum rather than a discrete stage of embryogenesis. Signalling pathways in pluripotent stem- maintenance A key goal of stem- research is to identify the factors that will enable researchers to propagate and differentiate pure populations of stem s, early lineage-committed progenitors and mature functional derivatives under defined conditions in vitro. It is widely thought that the same signals that regulate these processes in the peri-implantation embryo will control the maintenance of ES s in a pluripotent state in vitro and that knowledge of signalling pathways in mouse embryos can be harnessed in efforts to regulate human ES- differentiation 34. The evidence for this 34 notwithstanding, since the early studies of human pluripotent teratocarcinoma lines, there have been strong 715

4 INSIGHT REVIEW NATURE Vol June 2010 Activin, nodal, TGF-β BMP11, myostatin FGF2 NRG1 PDGF S1P SMAD2, SMAD3 Self-renewal Unknown factor MEK ERK cascade BMP2, BMP4, BMP7 Noggin GDF3 SMAD1, SMAD5, SMAD8 Nucleus NANOG Differentiation PI(3)K Neural Endodermal Viability AKT ES IGF2 Figure 2 Extrinsic signals that affect self-renewal, differentiation and viability of human ES s. Signalling mediated by members of the transforming growth factor-β (TGF-β) family such as TGF-β, activin and nodal, growth differentiation factors (GDFs, including myostatin) and bone morphogenetic proteins (BMPs) converges mainly on NANOG, which maintains ES s in an undifferentiated state with the ability to selfrenew. Signalling activity mediated by the MEK ERK receptor tyrosine kinase cascade allows self-renewal of ES s and maintains their viability (through inhibiting apoptosis and anoikis). In addition, insulin-like growth factor 2 (IGF2)-mediated signalling through phosphatidylinositol-3-oh kinase (PI(3)K) inhibits ES s from differentiating into endodermal lineage s. WNT-mediated signalling might affect these -fate decisions, but its role is controversial at present. NRG1, neuregulin 1; PDGF, platelet-derived growth factor; S1P, sphingosine 1-phosphate. suggestions that the extrinsic mechanisms that modulate stem- selfrenewal in humans were different from those in mice 35. Culture of the inner mass of the human blastocyst under the same conditions as those used to derive mouse ES s (that is, with feeder- support from embryonic fibroblasts and by supplementation with serum and LIF) gave rise to lines that expressed many of the same transcription factors that control pluripotency in mice. But these human lines differed considerably from mouse ES s in their expression of certain -surface markers and in their response to growth factors 1,2,36. However, as noted above, recent work has shown that several types of pluripotent stem can be isolated from the mouse embryo and that these types probably correspond to different stages of embryonic development. Thus, the differences between human and mouse ES s could be a consequence of species-specific differences in development, or it is possible that human and mouse ES s represent different stages of development. Among the most prominent features that discriminate between the populations of mouse pluripotent stem s are the extrinsic factors and the interular and intraular signalling systems that maintain the stem s. It is therefore instructive to examine the key signalling systems that maintain human ES s in their pluripotent state (Fig. 2) transforming growth factor-β (TGF-β), growth factors that signal through receptor tyrosine kinases (RTKs), WNTs, and LIF and JAK STAT noting the roles of these systems in mouse pluripotent stem- lines. TGF-β superfamily The TGF-β superfamily contains structurally related signalling proteins that mediate a broad range of biological effects through binding to -surface receptors. The superfamily includes the TGF-β proteins, activin and nodal, growth differentiation factors (GDFs) and BMPs, all of which are involved in maintaining the stem- state. Nodal is an important regulator of many processes in early embryonic development. Nodal and activin signal through the same receptors, but activin is often used as a surrogate factor for nodal in -culture experiments because of its wider availability as a recombinant protein and comparable activity. Activin and nodal have been shown to suppress the differentiation of human ES s 31,37,38. Consistent with this finding, human ES s express receptors for nodal (ACVR1B and ACVR2B) and a co-receptor for nodal (TDGF1; also known as cripto) 10. Interestingly, human ES s also express the nodal antagonists LEFTY1 and LEFTY2, as well as nodal itself. Various studies have shown that activin or nodal can synergize with several other extraular signalling proteins, more specifically FGF2 or WNTs, to promote stem- maintenance 31,37,39,40. One study found that when activin alone is added to human ES s cultured in serumfree medium, FGF2 is produced 39. Thus, activin does not directly support long-term human ES- maintenance but might do so at least in part by eliciting FGF2 production. The bulk of the evidence suggests that both signalling pathways (activin-mediated and FGF2-mediated) need to be activated for stem- maintenance. In addition, endogenous nodal-mediated signalling may be a key autocrine pathway of ES- maintenance: TGF-β can substitute for activin and/or nodal in human ES- maintenance, and blockade of the protein-kinase activity of the TGF-β receptor induces more rapid differentiation of human ES s than removal of exogenous TGF-β 41. The precise role of the nodal antagonists LEFTY1 and LEFTY2 in regulating stem- states is unclear. It is possible that these molecules are synthesized by a minor subpopulation of differentiated s 42. In the mouse blastocyst, Lefty1 is expressed by a few s in the inner mass 43,44 and later in a subset of the primitive endoderm and visceral endoderm s of the peri-implantation mouse embryo, suggesting that the Lefty1-expressing inner--mass s may be progenitors of endoderm s. In culture, LEFTY1 or LEFTY2 might therefore be produced by endoderm-like s, modulating the level of nodal-mediated signalling in the pluripotent s. Nodal has multiple roles in the mouse embryo 45. It is required for the maintenance and growth of the epiblast, as well as for sustaining the expression of pluripotency genes. Embryos that lack nodal lose expression of Oct4 (which encodes a pluripotency transcription factor) in the epiblast, and this is accompanied by premature differentiation into neuroectoderm. Later, nodal acts in conjunction with FGFs and WNT signals (both discussed below) to initiate differentiation into germ layers. The nodal- and activin-mediated signalling pathway activates the transcription factors SMAD2 and/or SMAD3, and expression of the key pluripotency transcription factor NANOG occurs downstream of this SMAD-mediated signalling in human ES s and mouse EpiSCs 23,41 (Fig. 2). Of all of the pluripotency actors, NANOG is downregulated the most rapidly after blockade of TGF-β- and/or activin-mediated signalling. SMAD2 and SMAD3 bind to the promoter of the gene encoding NANOG and activate its expression, whereas SMAD1, SMAD5 and SMAD8 (which are activated by BMPs) bind to the promoter and inhibit NANOG expression 716

5 NATURE Vol June 2010 REVIEW INSIGHT in human ES s. Activation of SMAD2- and/or SMAD3- mediated signalling or FGF2-mediated signalling suppresses BMP4 expression in human ES s 40, preventing spontaneous differentiation. Another set of TGF-β superfamily members, the GDFs, act through autocrine or paracrine pathways to maintain human ES- self-renewal. GDF3 is a member of the TGF-β superfamily related to Xenopus laevis vg1 (ref. 46). GDF3 is expressed by pluripotent populations in mice and humans 36, and has been shown in overexpression studies to antagonize BMPs and facilitate nodal activity in culture 47,48. In short-term assays, GDF3 supports maintenance of pluripotency marker expression in human ES s and blocks the BMP-mediated induction of differentiation 48. The enhancing effect of GDF3 on human ES- self-renewal might therefore be achieved through a combination of these two different activities on nodal- and BMP-mediated signalling, although a recent study strongly suggests that the physiological role of this protein might be to inhibit BMPs 49. Inhibition of BMP-mediated signalling and an appropriate balance between signalling involving SMAD2 and/or SMAD3 and that involving SMAD1, SMAD5 and/or SMAD8 are crucial for stem- self-renewal. It is possible that human ES s cultured in the presence of mouse embryonic fibroblasts (MEFs) are maintained in part by the activity of MEF-derived factors, such as the BMP antag onist GREM1 and TGF-β superfamily members (for example GDF11 and MSTN), which function by activating signalling through ACVR2B 50. As constituents of -culture supplements or as paracrine factors, BMPs have profound effects on human ES s. When these s are treated with BMPs, they differentiate into a variety of types. Antagonizing BMP-mediated signalling in human ES s can either enhance ES- self-renewal or drive ES s to adopt neural fates By contrast, in mouse ES s, BMPs function to maintain pluri potency 54, through inducing the expression of inhibitor of DNA binding (ID) proteins, which in turn suppress differentiation into neural types. This effect of BMPs and the ID proteins on stem- maintenance depends crucially on activating JAK STAT signalling by way of LIF, a pathway that does not operate in human ES s. In the mouse embryo, disruption of BMP-mediated signalling by deletion of Bmp4, Bmpr1a or Smad4 leads to reduced proliferation in the epiblast In summary, recent results highlight a key role for a balance between signalling mediated by SMAD2 and/or SMAD3 (driven by nodal and/ or activin) and signalling mediated by SMAD1, SMAD5 and/or SMAD8 (driven by BMPs) in human ES- maintenance. GDF3 might function at the intersection of these pathways, both driving self-renewal and blocking differentiation 41,46,58. RTK signalling mediated by growth factors The role of signalling through RTKs downstream of FGF2, insulin-like growth factors (IGFs) and platelet-derived growth factor (PDGF) also highlights differences between human and mouse ES s. FGF2 was the first factor found to be crucial for the maintenance of human ES s 59, and many chemically defined media incorporate this factor to enhance human ES- growth 59 (see ref. 60 for a review). Human ES s express receptors for FGFs and produce FGF2 (ref. 61), which activates signalling through the RTKs ERK1 and ERK2 in these s. Inhibition of this signal transduction pathway results in stem- differentiation 62,63. By contrast, in mouse ES s, activation of ERK1 and/ or ERK2 signalling leads to differentiation 64. The precise biological action of FGF2 on human ES s is unclear, although there is evidence that it maintains stem- phenotype, rather than promoting proliferation or inhibiting death. Other FGFs that signal through RTKs promote differentiation in mouse ES s, in contrast to their role in human ES s. In vitro, FGF4 drives the differentiation of ES s from FGF4-null mice into par ietal endoderm 65. FGFs can also support the growth of mouse tropho blast stem (TS) s 66, and the activation of RTK signalling represses the pluripotency transcription factor gene Nanog in mouse ES s 44,67. Conversely, blocking FGF4-mediated signalling by a chemical inhibitor or by abrogating downstream ERK1 and/or ERK2 activity allows mouse ES s to retain pluripotency and undergo self-renewal 68. FGF2 is presumed to act directly on human ES s, because its positive effect on stem- maintenance can be achieved in the absence of feeder s. A recent study, however, has challenged this assumption and suggests that FGF2 has a paracrine action 69. Under certain serum-free and feeder--free culture conditions, human ES s can differentiate into fibroblast-like s that seem to function as an autologous feeder layer for the stem s 70. These fibroblast-like s express a higher level of FGF receptors than do the undifferentiated human ES s in the same culture, and when treated with FGF2, they release IGF2. One of the key actions of IGFs could be to block activin- and/or nodal-mediated signalling, through activating phosphatidylinositol-3-oh kinase (PI(3)K) 71, which prevents the differentiation of human ES s into endoderm. Many human ES- systems routinely include the use of serum replacements, which may contain large amounts of insulin and thereby activate IGF receptors. The distinct effects of FGF2 are, however, apparent despite insulin supplementation. Thus, FGF2 may have direct effects on human ES s under conditions in which an autologous fibroblast feeder layer is not produced. Together with the lysophospholipid sphingosine 1-phosphate (S1P), PDGF (which signals through RTKs) can support the maintenance and survival of human ES s in the absence of serum 72,73. By contrast, mouse ES s do not express receptors for PDGF 74. The above findings about FGF2 and PDGF point to a role for RTK signalling in the maintenance of human ES s, in part through its effects on survival. This idea is further supported by a study showing that the RTK ERBB2 is expressed on ES s and that its ligand neuregulin 1 (NRG1) functions in ES- maintenance 75. An analysis of the phosphorylated proteome of human ES s also provides supporting evidence for important roles for several RTKs 73. This study showed that FGF2, PDGF, IGF2 and ERBB2 are involved in maintaining human ES- maintenance, confirming previous findings (noted above) on the role of these factors in human ES- maintenance. By contrast, there is no strong evidence that RTK signalling has a role in stem- maintenance in mice, with the exception of the survival effects of IGFs. One crucial issue to be resolved is how FGF2-mediated signalling and MEK ERK signalling (with MEKs being the protein kinase that activates ERKs) interact with the network of pluripotency transcription factors and activin- and/or nodal-mediated signalling to promote the self-renewal of stem s. There is some indication that activin- and/ or nodal-mediated signalling might be important for suppressing the action of FGF2 to induce differentiation into neural s 23, and several studies point to a role for signalling through the MEK ERK pathway and through the protein kinase AKT in preventing anoikis ( death on removal from an underlying substrate or extraular matrix) or apoptosis of human ES s 76,77. These findings do not, however, fully explain why FGF2 is required for stem- maintenance. WNT family The role of WNT-mediated signalling in the maintenance of human ES pluripotency remains uncertain. When the small molecule BIO was used to inhibit GSK3β a key component of the WNT-mediated signalling cascade (and of other signalling pathways, including those mediated by insulin and hedgehog) it was found to activate WNT-mediated signalling and allow short-term maintenance of human and mouse ES s in the undifferentiated state 78, as did treatment of the s with WNTs. Subsequent studies on human ES s indicate that the main effect of WNTs is to enhance proliferation 79, and additional factors such as TGF-β and FGF2 are required for long-term maintenance of stem s 31,39,80. The effects of activating the WNT pathway in ES s might be highly context dependent. A small molecule called IQ-1 has been shown to maintain mouse ES s in serum-free culture 81. Studies of the downstream effects of this molecule showed that the effect of WNT-mediated signalling in mouse ES s depends on the balance between the association of β-catenin with p300 and with CBP (also known as EP300 and CREBBP, respectively), two transcriptional co-activators involved in the nuclear effector response to WNT-mediated signalling. Treatment of mouse ES s 717

6 INSIGHT REVIEW NATURE Vol June 2010 XEN stem TS EG LIF, FCS and FGF2 PG FGF4, heparin and MEF-cm MEF-cm, FGF4 and LIF FGF4, MEF-cm and Cdx2 expression FGF2, activin and serum-free N2B27 medium BMP4, then noggin, chordin, activin and FGF2 Epiblast Blastocyst ES EpiSC FGF2 and activin res FGF2, activin and BIO MedII LIF and BMP4 FAB-SC EPL Activated LIF and JAK STAT signalling cascade, and trypsinization cepi Figure 3 Interconversion of mouse embryo-derived stem- types. Stem s with different characteristics can be derived from the mouse blastocyst or the early or late epiblast under different culture conditions. From the blastocyst, four types of stem have been harvested: ES s, TS s, XEN stem s and FAB-SCs. When FAB-SCs are cultured in medium supplemented with BMP4 and LIF, the s are converted into ES--like s. ES s can be converted into TS s by culturing them in mouseembryonic-fibroblast-conditioned medium (MEF-cm) containing FGF4, together with enforcing the expression of the transcription factor CDX2. ES s can be turned into EPL s by culturing them in MedII (medium conditioned by the human hepatocarcinoma line HepG2), and EPL s can be converted back into ES s by culturing them with LIF. Early (pre-gastrulation embryo) and late (gastrulating embryo) epiblast fragments give rise to EpiSCs when they are cultured in medium supplemented with FGF2 and activin. Single s from dissociated epiblasts that are cultured in MEF-cm with LIF and fetal calf serum (FCS) become cultured epiblast (cepi) s, which can be converted into reversed ES s (res s), which resemble ES s. EpiSCs can be converted into ES s by culturing them with LIF and inhibitors of GSK3β and ERKs or by enforced Klf4 expression. In the converse process, ES s can be turned into EpiSCs by culturing them with activin and FGF2. EpiSCs differentiate into s that resemble primordial germ (PG) s after being cultured with BMP4 and then with noggin and chordin in the presence of activin and FGF2. These PG s can be converted into pluripotent embryonic germ (EG) s by culturing them in medium supplemented with LIF, FCS and FGF2. with IQ-1 resulted in the association of β-catenin with p300, which is postulated to drive stem- maintenance at the expense of differentiation. It is not known whether this mechanism operates in human ES s, but it is possible that other extrinsic signals also converge on these transcriptional co-activators. In mice, there is no compelling evidence that active WNT-mediated signalling has a role in the maintenance of the pluripotent population in vivo, in the inner mass or the epiblast. Moreover, embryos that lack WNT3 activity show persistent expression of a pluripotency marker (Oct4) in the epiblast, which does not undergo germ-layer differentiation in these LIF LIF, and inhibitors of GSK3β and ERKs, or Klf4 expression LIF, FCS and MEF-cm (for single s) embryos 82. It is not known whether the loss of WNT3 in these embryos is compensated for by the activity of other WNTs that are expressed in periimplantation embryos 83. Nevertheless, the absence of WNT3-mediated signalling alone seems to relieve the epiblast from any influences that would induce it to embark on differentiation. LIF and JAK STAT activity LIF can activate JAK STAT signalling in human ES s, but this pathway (as noted above) does not maintain pluripotency in these s, which instead rely on FGF2-mediated ERK signalling. By contrast, mouse ES s can be maintained by LIF-mediated JAK STAT signalling. This signalling occurs upstream of pluripotency gene (Klf4, Sox2 and Oct4) activity and by way of a parallel molecular cascade, through AKT PI(3)K and MAP-kinase GRB2, and it regulates the activity of the transcription-factor-encoding gene Tbx3, which is upstream of NANOG activity 84. The action of LIF requires the presence of serum, which can be replaced by BMPs 85. Mouse ES s can also be maintained in a combination of small molecule inhibitors that block, in particular, the MAPkinase signalling pathway that leads to differentiation 86. Plasticity of embryo-derived stem s in vitro When human ES- cultures are analysed closely, it becomes apparent that they have a high degree of heterogeneity. Using flow cytometry, different subpopulations can be identified in these cultures, on the basis of their expression of stem- surface antigens. For example, in subpopulations of human ES s that were isolated according to their expression of the stem- antigen SSEA3, the clonogenic fraction (the fraction capable of forming colonies and therefore of self-renewal in vitro) resided in the SSEA3-expressing population, but s that did not express SSEA3 expressed many pluripotency genes such as NANOG or OCT4 at much higher levels than did fully differentiated s 87. Furthermore, on examining the expression of two pluripotency -surface markers, GCTM2 and CD9, by human ES s, the stem s were found to express a continuous quantitative spectrum of these -surface molecules rather than be segregated into expressing and non-expressing subpopulations Gene expression profiling of the fractionated s reveals that stem- -surface antigen expression reflects the level of expression of pluripotency genes, which may be correlated with the ability of s to form colonies in vitro. Moreover, single- transcript analysis clearly shows that the s with the greatest capacity to renew themselves express the highest level of pluri potency genes; the s at this end of the continuum can be thought of as being at the top of the pluripotency hierarchy. There is a progressively decreasing likelihood of self-renewal as the expression of stem- surface markers and pluripotency genes wanes. Many s co-express pluripotency genes and lineage-specific genes, but s at the top of the hierarchy are biased towards the expression of pluripotency genes. These studies suggest that the co-expression of lineage-specific and pluripotency genes, a property that human ES s have in common with mouse EpiSCs, is a feature of human ES s in the middle of the hierarchy; the s at the top are most likely to express pluripotency genes only and have the strongest capacity for self-renewal. The studies also show that the expression of some -surface molecules and secreted products was shut down before that of the pluripotency factors OCT4 or NANOG. In human ES s, TGF-β-mediated signalling occurs upstream of NANOG expression. The question of what ultimately regulates expression of these -surface receptors and secreted factors upstream of the canonical pluripotency transcription-factor network remains unanswered. Mouse ES- cultures are also heterogeneous, and mouse pluripotent stem s exist in several interconvertible states in vitro (Fig. 3). Conversion between the various stem- types can be induced by manipulating culture conditions, through adding or withdrawing certain cytokines and/or growth factors and through changing the transcriptional activity of genes (Fig. 2) that enforce lineage restriction. The ability to alter ular properties experimentally highlights the inherent plasticity of mouse stem s. Rathjen and co-workers were the first to demonstrate

7 NATURE Vol June 2010 REVIEW INSIGHT a reversible conversion of mouse ES s to a state (EPL s) that more closely resembled the early epiblast of the embryo. In another study, FAB-SCs, which are derived from the blastocyst but cannot form teratomas or chimaeras and are therefore not pluripotent, can be converted back to the ES- state by culturing for brief periods in the presence of LIF and BMP4 (ref. 21). Initial observations suggested that interconversion of mouse ES s and EpiSCs was possible only through manipulating expression of the transcription factor KLF4 (ref. 27), which is produced by ES s but not by EpiSCs, but more recent data (not shown in Fig. 3) show that mouse EpiSCs can be converted to ES s simply by culturing them in the presence of LIF, BMP4 and a feeder- layer 20. Moreover, by using appropriate reporter genes, s with the properties of the late epiblast, as well as other pluripotent types, can readily be detected in ES cultures 26,91. The facile interconversions of stem- types described here may reflect an underlying heterogeneity within mouse ES- and EpiSC populations, and possibly in other mouse stem- populations, although limited data are available for these. Outlook An emerging view of the stem- state holds that it is not an invariant and -autonomous state but, instead, should be considered as the dynamic response of the lineage as a whole to the external environment 92. The inner mass and epiblast of the mouse embryo, and presumably their human counterparts, are dynamic populations whose interactions with the extra-embryonic tissues surrounding them are crucial for -fate determination 9,34,93. These interactions evolve rapidly as the developmental program unfolds, and the response of the pluripotent s to external signals changes swiftly within a short time frame. This dynamism is reflected in the plasticity of pluripotent stem populations in vitro in response to manipulations of the -culture environment. 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