Abstract
The biology of embryonic stem (ES) cell lines has opened new avenues both in the biology of pathophysiological development and in potential regenerative medicine. The transforming growth factor (TGF)-β superfamily plays a major role in the development of organisms. The family comprises a variety of growth factors that feature disparate functions in the biology of ES cells. These factors regulate both stemness and various cell differentiation pathways. Despite intensive work, the role of this family of growth factors in the function of ES cells is still unclear. More specifically, mouse and human ES cells differentially respond to these factors. Inspired by the biology of development, this review summarizes the current knowledge on the pleiotropic effects of these growth factors on the fate of ES cells.
1. Introduction
Embryonic stem (ES) cells are derived from blastocysts, multicellular structures originating from four (human) to five (mouse) cleavages of fertilized oocytes. Isolated from the inner cell mass of blastocysts, the ES cells retain properties of self-renewal and the potential to be committed and to differentiate toward most cell lineages. They are thus able to spontaneously give rise to different progenies of the three embryonic layers, namely, the ectoderm, the mesoderm and the endoderm. Derivation of embryonic stem cell lines has opened new avenues both in biology of pathophysiological development and in potential regenerative medicine.
The transforming growth factor (TGFβ) superfamily plays a major role in the biology of development. TGFβ and associated members of the family are mostly pleiotropic growth factors. They are broadly expressed throughout the body and regulate many cellular pathophysiological processes including cell fate, cell proliferation, cell senescence, and tissue repair. Accordingly, these growth factors have been widely investigated in the biology of ES cells. Inspired by developmental biology, this review summarizes the current knowledge on the role of TGFβ superfamily members in both self-renewal and lineage commitment of mouse and human ES cells. It further explores the intracellular mechanisms underlying the action of TGFβ-related factors.
2 Intracellular signalling pathways of the TGFβ superfamily
To better understand the function of TGFβ superfamily members, their intracellular signalling pathways have to be well defined. Indeed, recent data about specificity of functions of Smads [1–4], which constitute the canonical signalling pathway of TGFβ superfamily, revealed a fine tuning regulation of this pathway and, in turn, of downstream transcriptional networks.
The family of TGFβ first discovered 25 years ago includes about forty polypeptidic growth factors that share similarities in their structure. It is classified into two groups: the first group includes the bone morphogenetic proteins (BMP) and the growth differentiation factors (GDF) and the second one comprises TGFβ, activin, and nodal. Three isoforms of TGFβ, TGFβ1, 2 and 3, and at least 20 BMPs and 15 GDFs have been described thus far. All these factors, whose expression and function have been conserved in many species throughout evolution, play a key role during embryonic development, in processes of cell differentiation [5].
The signalling pathways underlying the cellular function of the TGFβ superfamily members are several: Smad factors are the key mediators of the canonical TGFβ pathway. The TGFβ superfamily members bind to receptor complexes composed by TGFβ receptor type I, activin receptor I, and receptors type II (TGFβRII, BMP-R II, activin-RII), also called activin-like kinase receptor (Alk). These receptors feature two transmembrane glycoproteic domains which are able to dimerize through cystein residues following binding of a ligand. Upon agonist binding, the change in conformation of receptors induces activation of the serine/threonine kinase of RII that phosphorylates RI on specific serine and threonine residues present in the juxtamembrane glycine and serine-rich GS domain [6]. Phosphorylation-dependent activation of intracellular cytosolic mediators called Smads thus follows and leads to their nuclear translocation. This results in transcriptional activation and expression of target genes. This signalling pathway is regulated by cross-talks with other pathways including Wnt, Hedgehog or other tyrosine kinases-linked growth receptor signalling pathways. The intracellular signalling is tightly dependent upon the cell type or stage of cell differentiation. Smads are divided in two subgroups: the regulatory Smads 2/3, downstream of TGFβ-R, and 1,5,8, downstream of BMP, activin, and nodal receptors, and the integrative Smad4 which is the only one to bind DNA [6]. Smads feature a high specificity as to their targets and they work in a combinatorial manner [1–4]. This provides the cell with both a high specificity and a broad diversity in regulation of downstream transcriptional pathways. Smads include both NLS and NES sequences which allow these factors for shuttling between the cytosol and the nucleus. However, DNA binding of Smad4 is weak and requires co-factors (i.e. transcription factors) that also bind DNA to turn on gene transcription.
TGFβ superfamily members also activate MAPKs. Stimulation of TGFβ receptors turns on the activity of the mitogen-activated protein kinase kinase kinase (MAPKKK) TAK, acting via the p38 MAP kinase on the transcription factors CREB and ATF2. This pathway regulates many cellular processes including cardiac cell hypertrophy [7]. It is however unclear what the TGFβ-dependent MAPK signalling pathway regulates in ES cells. Ying et al. did not find any effect of BMP on p38 activation in mouse ES cells [8]. We also did not observe any effect of p38 or ERK inhibitors on the TGFβ signalling pathway in mouse ES cells [9].
3 Short overview of early cardiac development and role of the TGFβ superfamily: a model to direct the fate of ES cells
ES cells recapitulate the early stages of development. They represent an appropriate model to study the function of growth factors on cell differentiation. This research has thus been inspired by the biology of mouse development. Specifically, the heart is one of the first organs to form during embryogenesis. Myocardial precursors are present as early as in the posterior lateral region of the epiblast. They still express the POU transcription factor Oct-3/4 at early gastrulation [10], expression that predicts a role of this so called ES cell-specific transcription factor, in early mesodermal cardiogenic fate [9]. Then, at gastrulation, following ingression of the epiblast at the primitive streak, cardiac progenitors migrate from the anterior region of the primitive streak into the mesoderm. The first cells to ingress are mainly those of the heart. In fact, in the mesoderm, the spatial distribution of cardiac progenitors is mirrored by their location in the primitive streak. This suggests that the specification of epiblast cells to the mesodermal lineage is accomplished through a global control of the timing and pattern of morphogenetic movement in the course of gastrulation [10]. The whole morphogenetic process is under the control of growth factors, including those of the TGFβ superfamily.
Members of the TGFβ superfamily have been involved in induction of the mesoderm in Xenopus, zebrafish, chicken and mouse [11]. Dunn et al. [12] reported that loss of Smad3, an intracellular component of the TGFβ signalling pathway, impaired production of anterior axial mesendoderm, while selective ablation of both Smad2 and Smad3 from the epiblast disrupts specification of axial and paraxial mesodermal derivatives. The same authors further observed that Smad2/Smad3 double homozygous mutants lacked mesoderm. Thus, they demonstrated that dose-dependent Smad2 and Smad3 signals cooperatively mediate mesodermal specification in the early mouse embryo. Besides a direct regulation of specification of mesoderm, TGFβ potentiates FGF activity [13]. TGFβ is expressed early in the cardiac region of the mesoderm. BMPs also feature an important function in cardiogenesis. BMP-2 is expressed in the early embryo: added to the culture medium of embryos in vitro, the morphogen induces differentiation of the anterior median mesoderm between days 5 and 7 although this region is not a cardiogenic one [14]. In line with these data, BMP2 triggers ectopic expression of Nkx2.5, GATA-4, -5, and -6 [14]. BMP4 knockout mice fail to gastrulate and do not form mesoderm. BMPR1 and 2b receptor-deficient embryos also fail to form mesoderm [15].
These findings point out the crucial role of TGFβ superfamily members in directing a mesodermal fate during early embryogenesis. The biology of mouse development provided a rationale for investigating the function of TGFβ superfamily in early specification of ES cells.
4 Role of TGFβ superfamily in self-renewal of embryonic stem cells
Members of the TGFβ superfamily have been reported to play a role in both maintenance of self-renewal and lineage commitment of ES cells although their function is disparate. Indeed, the effect of TGFβ in stem cell renewal and lineage specification depends upon species, the cell line, and the timing. In mouse ES cells, BMP4, through Id proteins, but not TGFβ, was shown to sustain self-renewal in combination with LIF [8], in the absence of feeder cells or serum. This finding, if validated in human ES cells, is of importance to set up a culture of feeder-free ES cells for future clinical use. The effect of BMP looks specific to BMP/Smad1/5 although the reason for that specificity is unclear since TGFβ/Smad signalling is functional in both mouse [9] and human ES cells [16]. Nodal [17] and TGFβ [18] might also play a role in maintenance of human ES cell pluripotency when added at low concentration in the culture medium. The authors discussed that nodal might exert its effect by blocking neuroectodermal differentiation, a default differentiation pathway of ES cells. Nodal expression is maintained in human ES cells by Smad2/3-mediated activin signalling. FoxH1, a target of Smad2/3, regulates transcription of nodal and its antagonists Lefty A and B [16]. Recently, Xiao et al. reported that activin A (10ng/ml) was necessary and sufficient to maintain pluripotency of HES cells H1 and I6 cultured on matrigel. Activin exerts its action through inhibition of BMP signalling, which would rather promote differentiation of cells [19].
Yao et al. [20] also reported that activin prevented differentiation of HESC toward the trophoectoderm under feeder-free conditions and in a chemically defined medium. Altogether, these findings raise many questions as to the genuine function of the TGFβ superfamily in ES cell renewal. As with any pleiotropic factor, the effects of TGFβ superfamily members depend on their concentration as well as upon the presence of other factors. The identity of the latter is nevertheless difficult to uncover when ES cells are cultured on feeder cells, matrigel, and/or in medium containing serum or a serum replacement. Interestingly, a pioneering study by Yao et al. [20] showed that addition of BMP4 to their chemically defined medium containing activin for a long term (8–10 days) induces expression of cardiac markers in HESC cultured without feeders. This suggests that the combination of BMP and activin might constitute a cardiac induction cocktail. However this medium includes B27 medium, which contains bovine serum albumin that might trap many growth factors interfering with BMP signalling. More experiments performed under better defined and standardized conditions are still required to assess whether the differential effects of TGFβ superfamily members in mouse and human ES cells are only due to the species and not to the different culture conditions used. Furthermore, by recruiting different numbers of receptors, different concentrations of factors are likely to determine the strength of the intracellular cascade. This might lead to activation of specific transcriptional targets and, in turn, might affect specific cell functions. It is also worth pointing out that most of the experiments published thus far have been performed with a specific human ES cells line. Very few reports have compared the results obtained in different cell lines. As an example, nodal, which in combination with FGF can allow for the growth of the H9 cell line in the absence of feeder cells, is not able to support the propagation of HUES-1, -3, or -9 cell lines in the absence of feeder cells. Rather, it induces expression of mesodermal genes (our unpublished data) as recently reported [21].
5 Role of TGFβ superfamily in embryonic stem cells differentiation
5.1 Cardiac differentiation
Nodal, TGFβ, and BMP2 are all able to trigger expression of mesodermal (Brachyury, Tbx6…) and cardiac (Nkx2.5, Mef2c…) specific genes in mouse ES cells [22,9,23]. BMP4 also favours mesodermal specification of ES cells by upregulating brachyury in a concentration-dependent manner. It concomitantly blocks neuroectodermal differentiation [15]. BMP2-induced mesodermal and cardiac specification is translated into a full cardiogenic differentiation program leading to an enrichment of cardiomyocytes within embryoid bodies [22], a three dimensional structure including the three embryonic layers and recapitulating the early steps of embryogenesis. BMP2 switches on mesodermal (Tbx6) and cardiac (Nkx2.5) specific genes via Smad transcriptional activation [24,25]. Nanog, an ES cell-specific transcription factor underlying their pluripotency, blocks this signalling pathway by binding Smad1 [26].
The BMP-dependent cardiogenic effect was observed only if TGFβ or BMP2 was added to ES cells prior to differentiation or during the first two days of EB formation. The opposite effect was revealed if the factors were added later on to differentiating EBs [22]. These data emphasise the crucial importance of the time window of addition of the factor to obtain a specific cellular effect. Interestingly, expression and associated function of a factor in ES cells and derivatives often reflects the scenario that occurs in the embryo. As an example, the EGF–CFC molecule cripto, a partner in nodal signalling is first expressed in vivo in the inner cell mass of the blastocyst. Later on, it is found at E6.5 in the epiblast and in the primitive streak, in the mesoderm, and then at E8.5 in the cardiac region [27]. Cripto is also essential for cardiogenesis in EBs [28,29] and it turns out that cripto first expressed in ES cells but no longer in ES cell-derived cardiomyocytes is essential for cardiogenesis at a precise timing. Cripto acts mainly but not only by recruiting nodal at ALK 4 or 7 receptors and by further activating Smad2/3. The intracellular mediators, in combination with co-factors then switch on transcription of cardiac specific genes.
The first evidence for a role of both activin and TGFβ, in mesodermal commitment of human ES cells, was provided by Schuldiner et al. [30]. In contrast, Pera et al. [31] reported that BMP2 added at a concentration of 25 ng/ml to human ES cells gives rise to extra-embryonic endoderm, while Xu [32] showed that BMP2, 4, and 7 used at high concentration (100 to 300 ng/ml) rather induces differentiation of these cells toward throphoblasts. These findings reveal how difficult it is to assess the true effect of these factors when added in the presence of serum and FGF2 or in the presence of feeder cells that release many still unknown factors. A recent report, for example, showed that FGF2 used to support the propagation of HESC acts on feeder cells (mouse embryonic fibroblasts), switching on expression of members of the TGFβ superfamily such as BMP4, TGFβ1, Grem1, or Inhba [33]. In the absence of serum and FGF signalling, BMP2 used at a low concentration (5–10 ng/ml) strongly induced expression of mesodermal and cardiac-specific genes in both HES-1 and 3 (D. Melton cell lines) as well as in I6 (J. Itskovitz cell line) human cell lines both in undifferentiated ES cells and further in early EBs (manuscript in preparation). The effect of TGFβ superfamily members may however depend on the cell lines and on the timing of addition, which could explain the discrepancies observed in the literature. We observed, for example, a much stronger response to TGFβ of mouse BS1 ES cell line than of the CGR8 cell line [9].
TGFβ and related factors (BMP2, nodal) use Oct-4 as a mediator of their effect to commit mouse ES cells toward a mesodermal fate. Smad4 binds and transactivates Oct-4 promoter leading to upregulation of the transcription factor following ES cell stimulation with TGFβ1, BMP2 or nodal. SiRNA and cDNA antisense transfected into ES cells prevented both Oct-4 expression and mesodermal and cardiac specification of ES cells. In fact, in vivo Oct-3/4 is downregulated in the epiblast of nodal-deficient mice [34]. A similar downregulation was observed in transgenic mice lacking Smad2 [35]. This led to the absence of the mesodermal marker Brachyury suggesting that a TGFβ-related signalling pathway is required to maintain Oct-3/4 expression in the early embryo and that Oct-3/4 is required not only for self-renewal of cells but also for early mesodermal and cardiac cell commitment in the embryo [9].
Altogether, these data suggest a highly dynamic role for BMP in committing the fate of ES cells. The variety of cellular effect of TGFβ-related factors may depend on the environment surrounding the cells. The switch from self-renewal to differentiation upon addition of TGFβ related factors might be triggered by a combination of other signals induced by other factors (FGF, VEGF secreted by feeders or ES cells themselves, antagonists of TGFβ superfamily members) or cell–cell interactions. Furthermore the strength (i.e. factor concentration-dependent) of the intracellular signalling may affect the downstream transcriptional cascades. TGFβ superfamily members are likely to use different intracellular pathways (Id, Oct-4, Smad, MAPK…) to mediate different cell functions in ES cells.
GDF3 is one of the members of the TGFβ superfamily. It features 50% identity with BMP2 and 4. It is mammalian specific and is expressed as early as in undifferentiated mouse or human ES cells. Its expression decreases when cells differentiate. It is in fact an inhibitor of BMP pathway. GDF3 overexpressed in human ES cells contributes to the maintenance of stemness while reduced GDF3 in mouse ES cells in the absence of LIF prevent their normal differentiation. This highlights the fact that BMP is a differentiating agent in human ES cells while it would rather favour stemness in mouse ES cells cultured in the absence of LIF [36]. As found in human ES cells, BMP however remains a differentiating factor in mouse ES cells cultured in the presence of LIF to prevent methylation and in turn repression of Oct-4 promoter, a key element in the cardiogenic effect of BMP [9].
Lefty is also a component of the TGFβ superfamily signalling pathway. Lefty is crucial for mesendoderm development. It acts by antagonising nodal and other factors of the family including activin and BMP. Lefty exerts its function by antagonizing the EGF–CFC signalling pathway [37]. Lefty is regulated by Smad and is induced upon differentiation of ES cells. This factor is at the crossroad of stemness and differentiation process [38]. Dvash et al. [21] reported recently that it was transiently expressed in differentiating human ES cells which do not express Oct-4 any longer. They confirmed that lefty expression was regulated by nodal. Lefty turns out to be a very interesting factor whose expression might mark out early mesodermal progenitors. Thus, it still deserves more investigative work to better define its function in ES cell fate.
5.2 TGFβ and other cell fates
The TGFβ superfamily members are pleiotropic factors. Furthermore, the combination of TGFβ superfamily members with other growth factors affects their cellular effects. Besides cardiac lineage, these factors direct the fate of ES cells towards multiple cell lineages of the three embryonic layers although their functions are different in a murine or human context.
A few reports published these last years have revealed a function of TGFβ in directing the fate of mouse stem cells towards specific mesodermal lineages. It was first reported that TGFβ plays a role in smooth muscle differentiation of ES cells. Using a truncated TGFβRII as a dominant negative mutant expressed in mouse ES cells, Sinha et al. found that smooth muscle cell differentiation of ES cells requires endogenous TGFβ [39]. Differentiation of another cell component of vessels is regulated by TGFβ. The latent TGFβ1 binding protein (LTBP)-1 as well as TGFβ1 were both found as inducers of endothelial cell fate in ES cells [40]. Antibodies against LTBP-1 prevent endothelial cell formation within EBs. Similarly, antibodies raised against TGFβ added to EBs exert the same action while addition of TGFβ at low concentrations (1 ng/ml) to EBs improves endothelial differentiation.
BMP4 treatment of EBs induces expression of the erythroid markers EKFL and GATA1. BMP4 is however efficient only until day 4 of differentiation [41].
Endodermal lineages derived from mouse ES cells are also targets of the TGFβ superfamily. Specifically, TGFβ2 is capable of mimicking the effects of pancreatic rudiments and this effect was enhanced by cmix (a chick putative endoderm inducer gene) expression to trigger expression of the endodermal marker pdx1 [42]. TGFβ together with Hedgehog, retinoid acid, and FGF promote expression of pancreatic transcription factors in ES cell-derived embryoid bodies (EB) [43]. Overexpression of nodal in mouse ES cells induces upregulation of both mesodermal and endodermal cell markers while it also downregulates neuroectodermal markers [44].
Interestingly, TGFβ, applied to human ES cell-derived EBs at different time frames, prevents expression of endodermal, endothelial and hematopoietic markers, which contrasts with findings in the mouse in which TGFβ reduced the level of endodermal markers but increased endothelial marker expression. The authors interpreted such a difference between the two species by the different origins of the yolk sac hemangiogenic lineages in mouse and human embryos. The effects of TGFβ on the hypoblast, from which these cell lineages are derived in human, would decrease subsequent differentiation of hematopoietic, endothelial and endodermal cells. In contrast, TGFβ action on murine hypoblast, while affecting endoderm, would not affect the hemangiogenic lineages that are epiblast-derived in the mouse [45].
6 Open issues
It is now established that TGFβ superfamily members participate in cell fate decision of ES cells (Fig. 1). However, the role of TGFβ in cell cycle of ES cells has been poorly investigated. We found for example that some canonical targets of TGFβ such as cdk inhibitors are conserved in ES cells [9]. Undoubtedly, much more investigative effort has to be devoted to this important biological question. Recently, TGFβ has been reported to repress activity of the telomerase reverse transcriptase [46]; this issue should also be investigated in ES cells. The family features a variety of factors and the role of many of them in ES cells stemness or differentiation has not been investigated yet. It remains to be established what the specific role of each member of the family is and how they exert their action under controlled experimental conditions. The transcriptional cascade(s) switched on by Smad-specific signalling pathways is still missing in human ES cells. Uncovering the Smad cofactors as well as the target genes required to direct the fate of ES cells will advance the field of developmental cognitive research and of more clinically oriented research in human ES cells derived from genetically affected embryos.
Summary of the effects of the TGFβ superfamily in mouse and human ES cells. The size of the letters of factors is indicative of the different concentrations used to obtain a specific cell lineage.
References
Author notes
Time for primary review 24 days
