Extrinsic regulation of pluripotent stem cellsbiochemistry.okstate.edu/copy_of_file-prelim-pdfs-cohorts-2011... · Extrinsic regulation of pluripotent stem cells ... These cell types

1Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine, University of Southern California, Los Angeles, California 90033, USA. 2Embryology

Unit, Children’s Medical Research Institute, Westmead, New South Wales 2145, Australia. 3Discipline of Medicine, Sydney Medical School, University of Sydney, Sydney, New South Wales

2006, Australia.

Extrinsic regulation of pluripotent stem cells Martin F. Pera1 & Patrick P. L. Tam2,3

During early mammalian development, as the pluripotent cells 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-cell 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 cells in research and therapy.

Pluripotent stem cells 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 cells from the human embryo1,2 (human embryonic stem (ES) cells) and the development of pluripotent stem cells (induced pluri-potent stem (iPS) cells) through the reprogramming3 of adult human cells4,5 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 impor-tant not only for improving the understanding of mammalian embryo-genesis and cellular differentiation but also for developing successful stem-cell-based therapies for regenerative medicine.

Pluripotency is a transitory state of embryonic cells that exists only during a brief window of development. Shortly after the onset of embryo-genesis, the cells 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 mem-branes) or pluripotent progenitors, which form the three primary germ layers, from which all of the tissues of the fetus are formed. By contrast, stem cells derived from these embryonic cells can be maintained indefi-nitely in the pluripotent state in vitro. However, in mice, in which all of the stages of embryogenesis are experimentally accessible, different types of stem cell, with distinct phenotypes, have been derived from the embryo, suggesting that these cells 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 cell have a common genetic network of transcription factors that main-tains them in a pluripotent state, recent studies have shown that these cells have a highly plastic phenotype. Certain stem-cell types readily convert into other stem-cell types, and many types of stem cell and their descendants can interconvert in response to extracellular signals. The response to extrinsic signalling is crucial for understanding plasticity in pluripotent stem-cell populations, because extrinsic signals can be propagated through intracellular signal-transduction pathways that converge on the genetic network that controls pluripotency. In vivo, stem-cell plasticity might enable the body to compensate for cellu-lar loss or developmental delay in early embryogenesis, as well as to respond to the vastly changing demands for cell production during

tissue maintenance, remodelling, regeneration and repair in adults. In this Review, we discuss the multiple states of pluripotency in stem cells, as well as the signalling systems that maintain these states. We focus on human pluripotent stem-cell regulation and draw on relevant findings from studies of mouse stem cells. Recent surprising findings about how stem cells are regulated in both species are providing insight into the multiple states of pluripotency.

Pluripotent stem cells in mice and humansOver the past few years, studies by several groups have provided a clear definition of the molecular phenotype of human ES cells and have examined the relationship between human ES cells and mouse pluripotent stem cells in vitro and in the early embryo.

Molecular phenotype of human ES cellsHuman ES cells, like other stem-cell populations, are characterized by their developmental potential, transcriptional and epigenetic profiles, and cell-surface markers. Developmental potential is assessed by a bio-logical assay of the ability of the cell to give rise to all of the cell 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 cells. For mammalian stem cells, two tests have traditionally been used to assess pluripotency. When injected into ectopic sites in host animals, pluri-potent cells form benign growths called teratomas, which contain mul-tiple types of differentiated tissue representative of all three embryonic germ layers. Human ES cells can also be tested in this way. The defini-tive test for pluripotency, which is applicable only in animal models, is the generation of germline-competent chimaeras after the introduc-tion of stem cells into pre-implantation embryos that are subsequently allowed to develop to full term in foster mothers. The contribution of the stem cells to the chimaera not only reveals the extent to which cells can differentiate to form all of the body’s tissues but also provides proof of the functional capacity of the descendants of the stem cells. It remains untested whether pluripotent stem cells from non-human primates can participate in chimaera formation.

The characterization of cells 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 cells. More importantly, examination of the whole transcriptome, proteome and epigenome of human ES cells has provided insight into

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how pluripotency is regulated at the molecular level through common genetic networks6,7. Whereas many of the early studies of human ES-cell phenotype and gene expression were descriptive, recent molecular and genetic studies of pluripotent stem cells in vitro, and their counter-parts in the peri-implantation (around the time of implantation) embryo in mammals in vivo8,9, are leading to a better understanding of the mechanisms of pluripotency maintenance and lineage commit-ment in mouse and human ES cells. The human ES-cell transcriptome and epigenome have now been characterized: multiple data sets from microarray studies of human ES cells have recently been subjected to a meta-analysis10. Furthermore, a study of a large panel of stem-cell 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 cell lines of different origins11. This finding suggests that expression of these genes robustly defines a human ES cell.

A diverse and expanding set of cell-surface markers is used to charac terize human ES cells. 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-cell lines derived from human blastocysts, as well as iPS cells, and distinguishes these cells from the pluripotent stem-cell 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-cell lines, which were independently derived using a variety of techniques11 and are representative of diverse genetic backgrounds. Importantly, some of these markers are expressed in the inner cell mass of human blastocysts, suggesting that human ES cells have a similar surface phenotype to the pre-implantation epiblast, in the blastocyst12.

Mouse pluripotent stem cellsHow does the phenotype of human ES cells relate to that of mouse pluripotent stem-cell populations? There is a core set of transcription factors that functions to maintain the pluripotent state in all of these cell 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 pluri-potency genes in an active state and repressing the lineage-specific genes. This set of pluripotency-associated transcription factors is expressed in all pluripotent stem-cell types that have been studied so far in humans and mice. These cell types are distinguished by their expression of other genes, however (Table 1).

Many of the genes that are characteristic of human ES cells, such as OCT4 (ref. 13) and DNMT3B (which encodes a DNA methyltransferase)14, have been detected in the inner cell mass of human blastocysts, similarly to the cell-surface markers discussed above15. 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 cells most clearly correspond.

The mouse counterparts of these pluripotency transcription factor genes are also expressed in the pluripotent cells in the mouse embryo.

So far, three types of pluripotent cell and two types of lineage-restricted stem cell can be derived from early mouse embryos directly or through interconversion in vitro (Fig. 1). The pluripotent cell types are ES cells, early primitive ectoderm-like (EPL) cells16 and epiblast stem cells (EpiSCs)17,18. Another cell type, FAB-SCs, is nullipotent (unable to differentiate) but can be converted to pluripotency in vitro. The lineage-restricted stem-cell types are trophoblast stem (TS) cells and extra-embryonic endoderm (XEN) stem cells, 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 cells can be derived at a high frequency from the pre-implantation epiblast of the mouse blastocyst19. EPL cells16 are generated from ES cells 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 cells, they cannot participate in chimaera formation after injection into host blastocysts. EPL cells might be viewed as an inter-mediate between ES cells and EpiSCs, but a more direct comparison of these two cell types is warranted. Mouse EpiSCs are derived directly from the early epiblast, and their properties differ markedly from those of ES cells. EpiSCs can also be derived from the late epiblast (in the gastru-lating 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 cells have similar gene expression patterns to those of ES cells (Table 1). They are nullipotent but can attain the pluripotent state through conversion to an ES-cell-like intermediate21.

The properties of these mouse stem-cell types are thought to reflect their embryonic cell 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 cells are the in vitro equivalent of the epiblast of the pre-implantation embryo22. This stage of the epiblast is derived from the inner cell mass and appears after the formation of the first two extra-embryonic lineages, the trophectoderm and the primitive endo-derm. With some exceptions, the gene expression patterns and biologi-cal properties of mouse EpiSCs are broadly similar to those expected of the early post-implantation epiblast17,18. Cells of the late epiblast (of the gastru lating embryo), from which EpiSCs can also be generated20, 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 cell have allowed comparative analysis of mouse stem cells and human ES cells.

Human ES cells and their mouse counterpartsIt has been argued that human ES cells more closely resemble EpiSCs than the other pluripotent cell types derived from the mouse embryo. Indeed, some aspects of the growth requirements of human ES cells

Table 1 | Properties of various pluripotent cell populations grown in vitroType of stem cell Stem-cell 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 cells √ √ √ √ √ √ √ X √ X √ √ X* X √ √

Mouse EPL cells √ √ √ 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 cells √ √ √ √ √ √ √ X X √ √ X √ √ √ ND

Tick (√) means that the gene or cell-surface marker is expressed, the indicated growth factors are required for self-renewal, or the indicated cell type will form teratomas or participate in chimaera formation.

Cross (X) means that the gene or cell-surface marker is not expressed, the indicated factors are not required for self-renewal, or the indicated cell type will not form teratomas or participate in chimaera

formation. ND (not determined) means that the attribute was not examined. EpiSC, epiblast stem cell; EPL cell, early primitive ectoderm-like cell; FAB-SC, stem cell 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 cells in activin. †Cells grown in leukaemia inhibitory factor (LIF) revert to an ES-cell-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 cells.

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and EpiSCs in vitro (discussed in the next section) are similar and distinguish them from mouse ES cells. For example, both human ES cells and mouse EpiSCs17,18 require nodal or activin signals to maintain their pluripotent state, whereas mouse ES cells do not. Neither is main-tained in the pluripotent state by leukaemia inhibitory factor (LIF), which is required for maintaining mouse ES cells. Both grow poorly after dissociation into single cells, 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 cells23.

The similarity between EpiSCs and human ES cells makes for a com-pelling argument that they are counterparts, but there are crucial dif-ferences between the cell-surface marker and gene expression of the two cell 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 cells do not express SSEA1 but express alkaline phosphatase. Furthermore, in common with mouse ES cells, human ES cells 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 cells and EpiSCs. Dppa3 is activated on interconversion of mouse EpiSCs to ES cells26, and Klf4 overexpression can drive mouse EpiSCs to acquire ES-cell properties27. In addition, human ES cells express the gene encoding the transcrip-tion factor REX1 (also known as ZFP42)11,28, which is present in mouse ES cells but not EpiSCs, and they do not express the gene encoding the EpiSC marker FGF5 (refs 10, 11).

In addition to stem-cell gene and cell-surface marker expression, a close analysis of the growth requirements of the two cell types also places some caveats on the conclusion that human ES cells 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 cells do29. Moreover, responding to activin, which both of these cell types do, is not a definitive criterion for the pluripotent state. In this regard, one study has shown that activin could maintain ES cells derived from the mouse inner cell mass in serum-free culture30, 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-cell maintenance31.

It has been suggested that mouse EpiSCs and human ES cells 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, how-ever, that both cell types can give rise to both trophectoderm and extra-embryonic endoderm in vitro, a finding that is not easily reconciled with the idea that the cells represent an in vitro equivalent of the late mouse epiblast, which has lost the ability to form these lineages in vivo. In addi-tion, EpiSCs express transcription factors that are characteristic of early lineage commitment18. Human ES cells also express such factors, but this feature may be a characteristic of the heterogeneity of cell types in culture (see the section ‘Plasticity of embryo-derived stem cells 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 cell is the counterpart of the human ES cell. 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 impor-tant 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 tissues32,33. Finally, there is a high degree of heterogeneity in human ES-cell cultures (see the section ‘Plas-ticity of embryo-derived stem cells in vitro’), suggesting that these cell populations represent a developmental continuum rather than a discrete stage of embryogenesis.

Signalling pathways in pluripotent stem-cell maintenanceA key goal of stem-cell research is to identify the factors that will enable researchers to propagate and differentiate pure populations of stem cells, 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 cells in a pluripotent state in vitro and that knowledge of signalling pathways in mouse embryos can be harnessed in efforts to regulate human ES-cell differentiation34.

The evidence for this34 notwithstanding, since the early studies of human pluripotent teratocarcinoma cell lines, there have been strong

Figure 1 | Stem-cell types derived from mouse embryos around the time of implantation. At implantation, mouse blastocysts comprise three distinct cell types: the trophectoderm; the inner cell mass, which produces the primitive endoderm; and the ‘naive’ (or early pre-implantation) epiblast. Under appropriate in vitro culture conditions, three types of stem cell with a comparable potential for differentiation to each of the cell types of the blastocyst can be derived: ES cells, trophoblast stem (TS) cells and extra-embryonic endoderm (XEN) stem cells1–5,66,94–97. Another type of stem cell, FAB-SCs, can be obtained by culturing blastocysts in medium containing FGF2, activin and the GSK3β inhibitor BIO25. And a further stem-cell type, EPL cells, can be derived from ES cells by culturing them in a cell-line-conditioned medium21. TS cells and XEN stem cells 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 cells can be derived from the epiblast of the early post-implantation embryo at 5.5–5.75 days post coitum22,23. Like ES cells, these epiblast stem cells (EpiSCs) are pluripotent in that they differentiate into the full range of typical germ-layer tissues in vitro and into teratomas in vivo. Epiblast cells are progressively restricted in differentiation potential as they are allocated to the mesoderm and endoderm through cellular 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 cells of which have been experimentally shown by lineage analysis and fate-mapping studies to retain plasticity in cell fate.

Blastocyst Pre-gastrulation embryo Early gastrulation embryo

LateepiblastEarly

epiblast

Extra-embryonicectoderm

Trophectoderm

Visceralendoderm

Epiblast

Primitiveendoderm Primitive

streak

ES cells

EPL cells

FAB-SCs

TS cells

XEN cells

TS cells

EpiSCs

EpiSCs

Implantation Gastrulation

Extra-embryonicectoderm

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suggestions that the extrinsic mechanisms that modulate stem-cell self-renewal in humans were different from those in mice35. Culture of the inner cell mass of the human blastocyst under the same conditions as those used to derive mouse ES cells (that is, with feeder-cell support from embryonic fibroblasts and by supplementation with serum and LIF) gave rise to cell lines that expressed many of the same transcription factors that control pluripotency in mice. But these human cell lines differed con-siderably from mouse ES cells in their expression of certain cell-surface markers and in their response to growth factors1,2,36. However, as noted above, recent work has shown that several types of pluripotent stem cell can be isolated from the mouse embryo and that these cell types probably correspond to different stages of embryonic development. Thus, the dif-ferences between human and mouse ES cells could be a consequence of species-specific differences in development, or it is possible that human and mouse ES cells represent different stages of development.

Among the most prominent features that discriminate between the populations of mouse pluripotent stem cells are the extrinsic factors and the intercellular and intracellular signalling systems that maintain the stem cells. It is therefore instructive to examine the key signalling systems that maintain human ES cells in their pluripotent state (Fig. 2) — transform-ing 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-cell lines.

TGF-β superfamilyThe TGF-β superfamily contains structurally related signalling pro-teins that mediate a broad range of biological effects through binding to cell-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-cell 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 cell-culture experi-ments 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 cells31,37,38. Consistent with this finding, human ES cells express receptors for nodal (ACVR1B and ACVR2B) and a co-receptor for nodal (TDGF1; also known as cripto)10. Interest-ingly, human ES cells 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 extracellular signalling proteins, more specifically FGF2 or WNTs, to promote stem-cell maintenance31,37,39,40. One study found that when activin alone is added to human ES cells cultured in serum-free medium, FGF2 is produced39. Thus, activin does not directly sup-port long-term human ES-cell 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-cell maintenance. In addition, endogenous nodal-mediated signalling may be a key autocrine pathway of ES-cell maintenance: TGF-β can substitute for activin and/or nodal in human ES-cell maintenance, and blockade of the protein-kinase activity of the TGF-β receptor induces more rapid differentiation of human ES cells than removal of exogenous TGF-β41.

The precise role of the nodal antagonists LEFTY1 and LEFTY2 in regulating stem-cell states is unclear. It is possible that these molecules are synthesized by a minor subpopulation of differentiated cells42. In the mouse blastocyst, Lefty1 is expressed by a few cells in the inner cell mass43,44 and later in a subset of the primitive endoderm and visceral endoderm cells of the peri-implantation mouse embryo, suggesting that the Lefty1-expressing inner-cell-mass cells may be progenitors of endoderm cells. In culture, LEFTY1 or LEFTY2 might therefore be pro-duced by endoderm-like cells, modulating the level of nodal-mediated signalling in the pluripotent cells.

Nodal has multiple roles in the mouse embryo45. 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 expres-sion of Oct4 (which encodes a pluripotency transcription factor) in the epiblast, and this is accompanied by premature differentiation into neuro-ectoderm. 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 cells and mouse EpiSCs23,41 (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

Figure 2 | Extrinsic signals that affect self-renewal, differentiation and viability of human ES cells. 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 cells in an undifferentiated state with the ability to self-renew. Signalling activity mediated by the MEK–ERK receptor tyrosine kinase cascade allows self-renewal of ES cells 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 cells from differentiating into endodermal lineage cells. WNT-mediated signalling might affect these cell-fate decisions, but its role is controversial at present. NRG1, neuregulin 1; PDGF, platelet-derived growth factor; S1P, sphingosine 1-phosphate.

Self-renewal

Unknown

factor

SMAD2, SMAD3

Activin, nodal, TGF-β

BMP11, myostatin

MEK–ERKcascade

FGF2

NRG1

PDGF

S1P

Viability

PI(3)K IGF2

Neural

Differentiation

NANOG

Noggin GDF3

SMAD1, SMAD5, SMAD8

BMP2, BMP4, BMP7

Endodermal

AKT

ES cell

Nucleus

716




in human ES cells. Activation of SMAD2- and/or SMAD3- mediated signalling or FGF2-mediated signalling suppresses BMP4 expression in human ES cells40, preventing spontaneous differentiation.

Another set of TGF-β superfamily members, the GDFs, act through autocrine or paracrine pathways to maintain human ES-cell self-renewal. GDF3 is a member of the TGF-β superfamily related to Xenopus laevis vg1 (ref. 46). GDF3 is expressed by pluripotent cell populations in mice and humans36, and has been shown in overexpression studies to antag-onize BMPs and facilitate nodal activity in cell culture47,48. In short-term assays, GDF3 supports maintenance of pluripotency marker expression in human ES cells and blocks the BMP-mediated induction of differen-tiation48. The enhancing effect of GDF3 on human ES-cell self-renewal might therefore be achieved through a combination of these two differ-ent activities on nodal- and BMP-mediated signalling, although a recent study strongly suggests that the physiological role of this protein might be to inhibit BMPs49. Inhibition of BMP-mediated signalling and an appro-priate balance between signalling involving SMAD2 and/or SMAD3 and that involving SMAD1, SMAD5 and/or SMAD8 are crucial for stem-cell self-renewal. It is possible that human ES cells 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 ACVR2B50.

As constituents of cell-culture supplements or as paracrine factors, BMPs have profound effects on human ES cells. When these cells are treated with BMPs, they differentiate into a variety of cell types. Antag-onizing BMP-mediated signalling in human ES cells can either enhance ES-cell self-renewal or drive ES cells to adopt neural fates51–53. By con-trast, in mouse ES cells, BMPs function to maintain pluri potency54, through inducing the expression of inhibitor of DNA binding (ID) pro-teins, which in turn suppress differentiation into neural cell types. This effect of BMPs and the ID proteins on stem-cell maintenance depends crucially on activating JAK–STAT signalling by way of LIF, a pathway that does not operate in human ES cells. In the mouse embryo, disrup-tion of BMP-mediated signalling by deletion of Bmp4, Bmpr1a or Smad4 leads to reduced cell proliferation in the epiblast55–57.

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-cell maintenance. GDF3 might func-tion at the intersection of these pathways, both driving self-renewal and blocking differentiation41,46,58.

RTK signalling mediated by growth factorsThe 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 cells.

FGF2 was the first factor found to be crucial for the maintenance of human ES cells59, and many chemically defined media incorporate this factor to enhance human ES-cell growth59 (see ref. 60 for a review). Human ES cells express receptors for FGFs and produce FGF2 (ref. 61), which activates signalling through the RTKs ERK1 and ERK2 in these cells. Inhibition of this signal transduction pathway results in stem-cell differentiation62,63. By contrast, in mouse ES cells, activation of ERK1 and/or ERK2 signalling leads to differentiation64. The precise biological action of FGF2 on human ES cells is unclear, although there is evidence that it maintains stem-cell phenotype, rather than promoting proliferation or inhibiting cell death.

Other FGFs that signal through RTKs promote differentiation in mouse ES cells, in contrast to their role in human ES cells. In vitro, FGF4 drives the differentiation of ES cells from FGF4-null mice into par ietal endoderm65. FGFs can also support the growth of mouse tropho blast stem (TS) cells66, and the activation of RTK signalling represses the pluripotency transcription factor gene Nanog in mouse ES cells44,67. Con-versely, blocking FGF4-mediated signalling by a chemical inhibitor or by abrogating downstream ERK1 and/or ERK2 activity allows mouse ES cells to retain pluripotency and undergo self-renewal68.

FGF2 is presumed to act directly on human ES cells, because its posi-tive effect on stem-cell maintenance can be achieved in the absence of feeder cells. A recent study, however, has challenged this assumption and suggests that FGF2 has a paracrine action69. Under certain serum-free and feeder-cell-free culture conditions, human ES cells can differentiate into fibroblast-like cells that seem to function as an autologous feeder layer for the stem cells70. These fibroblast-like cells express a higher level of FGF receptors than do the undifferentiated human ES cells 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 signal-ling, through activating phosphatidylinositol-3-OH kinase (PI(3)K)71, which prevents the differentiation of human ES cells into endoderm. Many human ES-cell systems routinely include the use of serum replace-ments, 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 cells 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 cells in the absence of serum72,73. By contrast, mouse ES cells do not express receptors for PDGF74. The above findings about FGF2 and PDGF point to a role for RTK signalling in the maintenance of human ES cells, in part through its effects on cell survival. This idea is further supported by a study showing that the RTK ERBB2 is expressed on ES cells and that its ligand neuregulin 1 (NRG1) functions in ES-cell maintenance75. An analysis of the phosphorylated proteome of human ES cells also provides supporting evidence for important roles for sev-eral RTKs73. This study showed that FGF2, PDGF, IGF2 and ERBB2 are involved in maintaining human ES-cell maintenance, confirming previ-ous findings (noted above) on the role of these factors in human ES-cell maintenance. By contrast, there is no strong evidence that RTK signalling has a role in stem-cell 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 cells. 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 cells23, and several studies point to a role for signalling through the MEK–ERK pathway and through the protein kinase AKT in preventing anoikis (cell death on removal from an underlying substrate or extracellular matrix) or apoptosis of human ES cells76,77. These findings do not, however, fully explain why FGF2 is required for stem-cell maintenance.

WNT familyThe role of WNT-mediated signalling in the maintenance of human ES-cell pluripotency remains uncertain. When the small molecule BIO was used to inhibit GSK3β — a key component of the WNT-mediated signal-ling cascade (and of other signalling pathways, including those mediated by insulin and hedgehog) — it was found to activate WNT-mediated sig-nalling and allow short-term maintenance of human and mouse ES cells in the undifferentiated state78, as did treatment of the cells with WNTs. Subsequent studies on human ES cells indicate that the main effect of WNTs is to enhance proliferation79, and additional factors such as TGF-β and FGF2 are required for long-term maintenance of stem cells31,39,80. The effects of activating the WNT pathway in ES cells might be highly context dependent.

A small molecule called IQ-1 has been shown to maintain mouse ES cells in serum-free culture81. Studies of the downstream effects of this molecule showed that the effect of WNT-mediated signalling in mouse ES cells depends on the balance between the association of β-catenin with p300 and with CBP (also known as EP300 and CREBBP, respec-tively), two transcriptional co-activators involved in the nuclear effector response to WNT-mediated signalling. Treatment of mouse ES cells

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with IQ-1 resulted in the association of β-catenin with p300, which is postulated to drive stem-cell maintenance at the expense of differentia-tion. It is not known whether this mechanism operates in human ES cells, 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 cell population in vivo, in the inner cell 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

embryos82. 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 peri-implantation embryos83. 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 activityLIF can activate JAK–STAT signalling in human ES cells, but this path-way (as noted above) does not maintain pluripotency in these cells, which instead rely on FGF2-mediated ERK signalling. By contrast, mouse ES cells can be maintained by LIF-mediated JAK–STAT signal-ling. 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 activity84. The action of LIF requires the presence of serum, which can be replaced by BMPs85. Mouse ES cells can also be maintained in a combi-nation of small molecule inhibitors that block, in particular, the MAP-kinase signalling pathway that leads to differentiation86.

Plasticity of embryo-derived stem cells in vitroWhen human ES-cell cultures are analysed closely, it becomes apparent that they have a high degree of heterogeneity. Using flow cytometry, dif-ferent subpopulations can be identified in these cultures, on the basis of their expression of stem-cell surface antigens. For example, in subpopu-lations of human ES cells that were isolated according to their expression of the stem-cell 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 cells that did not express SSEA3 expressed many pluripotency genes such as NANOG or OCT4 at much higher levels than did fully differentiated cells87. Furthermore, on exam-ining the expression of two pluripotency cell-surface markers, GCTM2 and CD9, by human ES cells, the stem cells were found to express a con-tinuous quantitative spectrum of these cell-surface molecules rather than be segregated into expressing and non-expressing subpopulations88–90. Gene expression profiling of the fractionated cells reveals that stem-cell cell-surface antigen expression reflects the level of expression of pluri-potency genes, which may be correlated with the ability of cells to form colonies in vitro. Moreover, single-cell transcript analysis clearly shows that the cells with the greatest capacity to renew themselves express the highest level of pluri potency genes; the cells 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-cell surface markers and pluripotency genes wanes. Many cells co-express pluripotency genes and lineage-specific genes, but cells 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 cells have in common with mouse EpiSCs, is a feature of human ES cells in the middle of the hierarchy; the cells at the top are most likely to express pluripotency genes only and have the strongest capacity for self-renewal. The stud-ies also show that the expression of some cell-surface molecules and secreted products was shut down before that of the pluripotency fac-tors OCT4 or NANOG. In human ES cells, TGF-β-mediated signalling occurs upstream of NANOG expression. The question of what ultimately regulates expression of these cell-surface receptors and secreted factors upstream of the canonical pluripotency transcription-factor network remains unanswered.

Mouse ES-cell cultures are also heterogeneous, and mouse pluripotent stem cells exist in several interconvertible states in vitro (Fig. 3). Conver-sion between the various stem-cell types can be induced by manipulating culture conditions, through adding or withdrawing certain cytokines and/or growth factors and through changing the transcriptional activ-ity of genes (Fig. 2) that enforce lineage restriction. The ability to alter cellular properties experimentally highlights the inherent plasticity of mouse stem cells. Rathjen and co-workers were the first to demonstrate16

Figure 3 | Interconversion of mouse embryo-derived stem-cell types. Stem cells 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 cell have been harvested: ES cells, TS cells, XEN stem cells and FAB-SCs. When FAB-SCs are cultured in medium supplemented with BMP4 and LIF, the cells are converted into ES-cell-like cells. ES cells can be converted into TS cells by culturing them in mouse-embryonic-fibroblast-conditioned medium (MEF-cm) containing FGF4, together with enforcing the expression of the transcription factor CDX2. ES cells can be turned into EPL cells by culturing them in MedII (medium conditioned by the human hepatocarcinoma cell line HepG2), and EPL cells can be converted back into ES cells 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 cells from dissociated epiblasts that are cultured in MEF-cm with LIF and fetal calf serum (FCS) become cultured epiblast (cEpi) cells, which can be converted into ‘reversed’ ES cells (rES cells), which resemble ES cells. EpiSCs can be converted into ES cells by culturing them with LIF and inhibitors of GSK3β and ERKs or by enforced Klf4 expression. In the converse process, ES cells can be turned into EpiSCs by culturing them with activin and FGF2. EpiSCs differentiate into cells that resemble primordial germ (PG) cells after being cultured with BMP4 and then with noggin and chordin in the presence of activin and FGF2. These ‘PG’ cells can be converted into pluripotent embryonic germ (EG) cells by culturing them in medium supplemented with LIF, FCS and FGF2.

cEpicell

XENstem cell

Blastocyst

LIF and BMP4

FGF2, activin

and BIO

Activated LIF

and JAK–STAT

signalling cascade,

and trypsinization

Epiblast

EScell

EpiSC

EPLcell

TScell

FAB-SC

‘PG’cell

EGcell

rEScell

FGF4, MEF-cm and

Cdx2 expression

LIF, FCS

and

FGF2

MedII

LIF

LIF, and inhibitors

of GSK3β and ERKs,

or Klf4 expression

LIF, FCS and

MEF-cm (for

single cells)

FGF2 and activin

FGF2, activin

and serum-free

N2B27 medium

BMP4, then

noggin, chordin,

activin and FGF2

FGF4, heparin

and MEF-cm

MEF-cm,

FGF4 and LIF

E

F

Epiblast

GF2 and activin

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a reversible conversion of mouse ES cells to a state (EPL cells) that more closely resembled the early epiblast of the embryo. In another study, FAB-SCs, which are derived from the blastocyst but cannot form terato-mas or chimaeras and are therefore not pluripotent, can be converted back to the ES-cell state by culturing for brief periods in the presence of LIF and BMP4 (ref. 21). Initial observations suggested that interconversion of mouse ES cells and EpiSCs was possible only through manipulating expression of the transcription factor KLF4 (ref. 27), which is produced by ES cells but not by EpiSCs, but more recent data (not shown in Fig. 3) show that mouse EpiSCs can be converted to ES cells simply by culturing them in the presence of LIF, BMP4 and a feeder-cell layer20. Moreover, by using appropriate reporter genes, cells with the properties of the late epi-blast, as well as other pluripotent cell types, can readily be detected in ES-cell cultures26,91. The facile interconversions of stem-cell types described here may reflect an underlying heterogeneity within mouse ES-cell and EpiSC populations, and possibly in other mouse stem-cell populations, although limited data are available for these.

OutlookAn emerging view of the stem-cell state holds that it is not an invariant and cell-autonomous state but, instead, should be considered as the dynamic response of the cell lineage as a whole to the external envir-onment92. The inner cell mass and epiblast of the mouse embryo, and presumably their human counterparts, are dynamic cell populations whose interactions with the extra-embryonic tissues surrounding them are crucial for cell-fate determination9,34,93. These interactions evolve rapidly as the developmental program unfolds, and the response of the pluripotent cells to external signals changes swiftly within a short time frame. This dynamism is reflected in the plasticity of pluripotent stem-cell populations in vitro in response to manipulations of the cell-culture environment. Although it is well known that mammalian embryos show regulative development (in which damage or loss of cells triggers com-pensatory mechanisms so that the embryo can develop normally), the mechanisms that enable pluripotent and extra-embryonic cell popu-lations to respond flexibly to changes in their environment have not been well defined. Heterogeneity and plasticity are both features of in pluripotent stem-cell populations that could be exploited to aid in the propagation and directed differentiation of cells in vitro, as well as in refining strategies for cell reprogramming. ■

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www.nature.com/reprints. The authors declare no competing financial interests.

Correspondence should be addressed to M.F.P. ([email protected]) or P.P.L.T.

([email protected]).

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