Placenta
Volume 31, Issue 11 , Pages 944-950, November 2010

Regulation of early trophoblast differentiation – Lessons from the mouse

  • C.E. Senner

      Affiliations

    • Laboratory for Developmental Genetics & Imprinting, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK
    • Centre for Trophoblast Research, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK
    • ,
  • M. Hemberger

      Affiliations

    • Laboratory for Developmental Genetics & Imprinting, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK
    • Centre for Trophoblast Research, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK
    • Corresponding Author InformationCorresponding author. Laboratory for Developmental Genetics & Imprinting, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK. Tel.: +44 1223 496 534; fax: +44 1223 496 022.

Accepted 26 July 2010. published online 27 August 2010.

Article Outline

Abstract 

The earliest stages of trophoblast differentiation are of tremendous importance to mediate implantation and to lay the anatomical foundations for normal placental development and function throughout gestation. Yet our molecular insights into these early developmental processes in humans have been limited by the inaccessibility of material and the unavailability of trophoblast cell lines that fully recapitulate the behaviour of early placental trophoblast. In this review we highlight recent advances that have come from the study of distinct stem cell types representative of the embryonic and extraembryonic lineages in the mouse, and from the study of mouse mutants. These models have revealed the presence of intricate transcriptional networks that are set up by signalling pathways, translating extracellular growth factor and cell positional information into distinct lineage-specific transcriptional programmes. The trophoblast specificity of these networks is ensured by epigenetic mechanisms including DNA methylation and histone modifications that complement each other to define trophoblast cell fate and differentiation. Despite the anatomical differences between mouse and human placentas, it seems that important aspects of early trophoblast specification are conserved between both species. Thus we may be able to build on our insights from the mouse to better understand early trophoblast differentiation in the human conceptus which is important for improving assisted reproductive technologies and may enable us in the future to derive human trophoblast stem cell lines. These advances will facilitate the investigation of genetic, epigenetic and environmental influences on early trophoblast differentiation in normal as well as in pathological conditions.

Keywords: Transcriptional networks, Epigenetic regulation, Trophoblast stem cells, Cell lineages, Hippo signalling, Ras–Mapk signalling

 

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1. Introduction 

The first differentiation event in mammalian embryonic development gives rise to two distinct cell populations; the trophectoderm (TE) which will go on to form all trophoblast cell types of the placenta, and the inner cell mass (ICM) which will give rise to the embryo proper. This divergence in cell fate is morphologically obvious at the blastocyst stage where the TE consists of a layer of epithelial cells surrounding the fluid-filled blastocoel cavity and the ICM. However, the cascade of events that leads to the separation of these first two cell lineages is initiated much earlier in the blastomeres of the early mouse embryo, and occurs in close interplay between transcription factors, epigenetic modifiers and signalling pathways as well as cell positioning and polarization events [1]. Thus cells of the 8-cell embryo become morphologically polarized with a microvillous apical membrane and an uneven distribution of specific proteins at either the apical (e.g., atypical protein kinase C (aPKC) and its complex component Par3) or basolateral (e.g., the Par1 homologue Emk1) pole [2]. Subsequent cell divisions in the transition to 16- and 32-cell embryos can then either produce two equally polarized cells (symmetric cell division); or, alternatively, position of the cleavage plane perpendicular to the apical–basal axis can generate one cell that is sent inwards and loses its polarity while the other daughter remains on the outside. Crucially, the daughters of such asymmetric divisions are not only spatially distinct but also inherit different molecular components. These events lead to the formation of apolar ‘inside’ cells and polar ‘outside’ cells whose cell fates are biased towards the ICM and TE, respectively, as the morula undergoes cavitation to form a blastocyst at embryonic day 3.5 (E3.5). Perturbation of cell polarity can affect cell position and consequently has an important role in the first cell fate determination event [3].

After implantation of the blastocyst, TE cells overlying the ICM (polar TE) proliferate in response to Fgf4 that is selectively expressed and secreted by the ICM, while TE cells not in contact with the ICM (mural TE) differentiate into primary trophoblast giant cells. The polar TE-derived cells go on to form the extraembryonic ectoderm (ExE) and ectoplacental cone (EPC). Stem cell potential is maintained in trophoblast cells of the ExE post-implantation. This is reflected by the ability to derive morphologically and functionally indistinguishable trophoblast stem (TS) cell lines from blastocyst stage embryos as well as from the ExE and its derivatives in the chorion until E8.5 [4], [5]. In addition to TE-derived TS cells, the blastocyst stage embryo can give rise to embryonic stem (ES) cells that are derived from the ICM. Lineage identity is fully committed in these stem cell types such that in chimeric embryos, ES cells predominantly contribute to the embryo and TS cells only contribute to the various trophoblast cell types of the placenta. Along with studies of mouse mutants these stem cell lines have allowed us to begin to elucidate the transcriptional networks that define the two earliest cell populations and orchestrate lineage-specific transcriptional programmes in all their progenitor cells [6]. But how does the compacted morula translate information on polarity and position into a transcriptional programme? And how does this transcriptional programme regulate the development of the trophoblast compartment? In this review we discuss the recent advances in our understanding of how signalling pathways establish the transcriptional circuitry that underpins TE identity and how the core trophoblast transcription factors coordinate lineage commitment, maintenance of the stem cell niche and eventual differentiation into placental cell types. In light of these recent findings we also re-examine the identity of trophoblast-like cells that have been derived from human ES (hES) cells and suggest key features expected of human trophoblast stem (hTS) cells that may facilitate the establishment of ‘true’ hTS cell lines.

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2. Transcriptional networks in early mouse trophoblast and TS cells 

One of the best-studied determinants of trophoblast cell fate is the caudal-type homeobox gene Cdx2 [7]. Cdx2 is required to restrict expression of the pluripotency factors Oct4 and Nanog to the ICM and, although an early blastocyst can initially be formed in its absence, Cdx2 is crucial to maintain a functional TE. It was found that Cdx2 and Oct4 have mutually antagonizing functions in early lineage formation such that Cdx2 over-expression as well as Oct4 knockdown in ES cells leads to transdifferentiation into TS-like cells [8]. Despite this central role of Cdx2 and Oct4 as reciprocally inhibitory transcription factors in lineage specification, the factor currently on top of the TE fate-determining hierarchy is Tead4 [9], [10], [11]. Tead4 is required earlier than Cdx2 as Tead4 mutants die before blastocyst formation and lack expression of Cdx2. In ES cells, over-expression of constitutively active Tead4 also leads to the derivation of TS-like cell lines [11]. Hence Tead4 is positioned upstream of Cdx2 and promotes trophoblast cell fate through both Cdx2-dependent and -independent mechanisms.

In addition to the early-acting roles of Tead4 and Cdx2 in lineage specification, several other transcription factors are expressed in the TE lineage and/or TS cells that do not appear to be required for lineage specification at the blastocyst stage, but rather for the further expansion of the stem cell niche from the polar TE that forms the ExE (Fig. 1). Eomes is one such factor, expressed in the TE only from the blastocyst stage onwards [12] and acting later in TE differentiation than Tead4 and Cdx2 by enhancing Cdx2 expression and promoting the expansion of the ExE [7], [13]. Ets2, Tcfap2c (AP-2γ) and Elf5 seem to have a similar role [14], [15], [16], [17], [18]. Although mutants of these factors develop slightly further than Eomes−/− embryos, ExE formation is severely impaired, TS cells cannot be derived from mutant blastocysts, and expression of core TS cell transcription factors such as Eomes and Cdx2 is significantly reduced. The timing of their expression and the similarity in their mutant phenotypes suggest that these factors act in the polar TE and ExE in response to the Fgf4 signal emanating from the adjacent ICM/epiblast to maintain a stem cell population required for continued development of this trophoblast tissue and to reinforce the lineage-specific transcriptional programme. Therefore Ets2, Tcfap2c and Elf5 can also be considered key players in the trophoblast/TS cell transcriptional network [19], [20], [21] (Fig. 1).

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  • Fig. 1 

    Transcriptional regulation of trophoblast lineage identity. (A) The interaction of key trophoblast transcription factors can be divided into three distinct stages. Tead4 triggers, directly or indirectly, the expression of Cdx2 and other transcription factors [9], [10], [11]. Once specified, a positive feedback loop involving Cdx2, Eomes, Tcfap2c, and Elf5 reinforces trophoblast identity [17], [20], [21]. In addition to supporting this network, Gata3, Elf5 and Ets2 subsequently act to drive further differentiation of the lineage into different placental cell types [19], [21], [22], [23], [24]. The dashed lines indicate presumptive, albeit not yet directly proven, interactions between Eomes, Gata3, Elf5 and Ets2 with Tcfap2c. Eomes can be activated directly by Elf5 and Tcfap2c, and directly or indirectly by Cdx2; the effect of other transcription factors on Eomes is yet to be tested [12], [20], [21]. (B) Timing of expression of key trophoblast transcription factors throughout trophoblast differentiation, and (C) pattern of expression in the early post-implantation (E6.0–E6.5) mouse embryo, as indicated by grey shadings. Tead4 is the earliest gene detected at the 4-cell stage by RT-PCR; Cdx2, Tcfap2c and Gata3 are expressed from around the 8-cell stage onwards [7], [10], [20], [23], while Eomes is detected only after the 16-cell stage and co-localises with the other transcription factors in the trophectoderm of the blastocyst [12]. Ets2 and Elf5 (mRNA and/or protein) are not detected at the early blastocyst stage, but are activated around E4.5–E5.0 and are expressed in the trophoblast compartment immediately after implantation [14], [17], [18], [21]. Co-expression of these transcription factors in the proximal ExE defines the niche of trophoblast cells with stem cell potential. E = embryo; ExE = extraembryonic ectoderm; EPC = ectoplacental cone.

Consistent with such a central role in defining trophoblast identity, a recent study has shown that over-expression of Tcfap2c in ES cells leads to the expression of other core trophoblast transcription factors including Elf5 and Cdx2 and the establishment of stable TS-like cell lines. Tcfap2c has Cdx2-dependent and -independent functions and can induce trophoblast cell fate even in a Cdx2 null background. Similarly, trophoblast fate induced by Cdx2 does not require Tcfap2c. However, activation of Elf5 is only achieved in the presence of both factors [20], suggesting that Tcfap2c and Cdx2 act in alternate pathways and are both required for the full establishment of TS cell identity.

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3. Regulation of the TS cell transcriptional circuit 

A key criterion by which a role of these various transcription factors in trophoblast determination and/or commitment of trophoblast lineage fate has been determined is their ability to induce trophoblast ‘trans’differentiation when ectopically expressed in ES cells. Interestingly, this assessment method has revealed subtle differences in the precise function of these genes, dividing them into two groups (Fig. 1): Tead4, Cdx2, Tcfap2c and presumably Eomes induce a lineage fate switch when over-expressed in ES cells, converting them into self-renewing TS cell-like cell lines [8], [11], [20]. By contrast, over-expression of Elf5, Ets2 as well as another transcription factor important for ExE expansion, Gata3, induces trophoblast cell fate from ES cells but at the same time triggers trophoblast differentiation into post-mitotic cells, such that stable TS cell lines cannot be derived [21], [22], [23], [24]. Thus, as judged by this differentiation-promoting role, Elf5, Ets2 and Gata3 are functionally distinct from the other transcription factors. This functional difference may balance the requirements for maintenance of the stem cell niche and the differentiation into cells of the nascent placenta. Because of the mutual transcriptional activation of these factors, this scenario also implies a self-regulatory limitation to the persistence of trophoblast stem cells in vivo. Thus, concomitant activation of differentiation-promoting factors by the TS cell transcriptional circuit ultimately pushes cells with stem cell potential out of the stem cell niche. This auto-regulation of trophoblast (stem) cell proliferation may represent a vital control mechanism to prevent aggressive tumour formation in the maternal uterus. It also suggests that TS cells are in a very labile state between self-renewal and differentiation, which may be reflected by the constant level of spontaneous differentiation that is consistently observed in TS cell cultures in vitro even in conditions that favour stem cell maintenance.

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4. Epigenetic regulation of lineage-specific transcriptional programmes 

Of all these various trophoblast transcription factors, Elf5 appears to have a special role as its epigenetic regulation by DNA methylation positions it as a gatekeeper of cell lineage fate between the trophoblast and embryonic compartments. Elf5 expression is initiated from the late blastocyst stage onwards in the ExE where it is important to maintain the expression of Cdx2 and Eomes [17], [21]. Consistent with its expression in trophoblast cells, the Elf5 promoter is unmethylated in TS cells, but methylated in ES cells where Elf5 is not expressed [21]. This differential epigenetic modification of Elf5 establishes a stable cell lineage barrier between the embryonic and trophoblast compartments as it restricts the positive transcriptional feedback loop between Cdx2, Eomes and Elf5 to the trophoblast lineage.

In addition to the stable repression of Elf5 by DNA methylation, complementary epigenetic mechanisms contribute to the maintenance of lineage identity in the early embryo and in derived stem cells. The asymmetric distribution of several histone modifications as well as their modifying enzymatic complexes between TE and ICM, and TS and ES cells, indicates that alternative mechanisms are utilised in a lineage specific manner to regulate gene expression programmes [6], [25]. Most recent studies have specifically highlighted the importance of the repressive histone H3 lysine 9 trimethylation (H3K9me3) mark for silencing of lineage-inappropriate genes. Many developmental regulators that are not expressed in pluripotent cells (ICM and ES cells) but that are required in later embryonic development are characteristically marked by both active (H3K4me2/3) and repressive (H3K27me3) histone modifications [26], [27], [28], priming them for activation. In trophoblast, however, where these genes are not required, this ‘poised’ state of bivalency is resolved by addition of H3K9me3 as well as DNA methylation, and/or by loss of H3K27me3, a modification that is globally under-represented in extraembryonic lineages [29], [30], [31]. It seems that the H3K9me3 modification at these sites is mediated by recruitment of the H3K9 methyltransferase Suv39h1 that is highly expressed in the trophoblast lineage [30]. The resulting trivalent (H3K4me2/3, H3K27me3, H3K9me3) or distinct type of bivalent (H3K4me3, H3K9me3) histone configuration mediates the repression of embryo-specific genes in the trophoblast compartment.

Recent studies have also revealed the involvement of another H3K9 methyltransferase, Eset, in repression of aberrant transcriptional programmes, notably that of trophoblast genes, in ES cells and the ICM [32], [33], [34]. Eset can interact with Oct4 and thereby confer transcriptional repressor functions to this transcription factor. Thus, Oct4 binds to and recruits Eset to trophoblast genes, leading to their epigenetic silencing by histone H3K9 methylation. Consequently, depletion of Eset in ES cells leads to the expression of TS cell genes such as Cdx2 and Tcfap2a and the formation of trophoblast-like colonies. However, Eset depletion only activates a partial trophoblast transcriptional programme and does not allow the derivation of stable TS cell lines, consistent with Elf5 remaining fully methylated and repressed. Further to this, Eset knockdown embryos can still form blastocysts, indicating that Eset is not required for the initial cell fate decision but rather contributes to the stable repression of trophoblast genes in cells of the embryonic lineage thereafter. What is not clear is how Eset is recruited in such a discriminate way to some Oct4 targets and not others. It has been reported that the Oct4–Eset interaction is dependent on SUMOylation of Oct4 [33]. Clearly other proteins are involved but the key players and mechanisms remain to be elucidated. However, collectively, these recent studies have revealed that the distinction between embryonic and trophoblast cell fates is achieved by different epigenetic mechanisms that function in parallel pathways: (i) by DNA methylation imposing a stable epigenetic repression of Elf5; (ii) by OCT4/ESET providing temporary and reversible silencing of other trophoblast factors including Cdx2, Eomes and Tcfap2a in cells of the embryonic cell lineage; and (iii) by resolution of H3K4me3/H3K27me3 bivalency-mediated gene priming through H3K9 methylation and loss of H3K27me3 to repress embryonic genes in the trophoblast compartment.

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5. Signalling pathways 

Although the mechanisms of lineage determination in the early embryo have been studied in substantial detail, a key question that remains is how the initial bias in expression of lineage-defining factors is triggered. This is particularly highlighted by the fact that Tead4 is ubiquitously expressed in all blastomeres, raising the question; how can it selectively regulate transcription in presumptive TE cells? To gain further insights into the question how the distinct transcriptional programmes are set up in the emerging two cell lineages, it is necessary to look at positioning and polarity events that generate inside and outside cells and the signalling pathways upstream of the transcriptional regulators.

In mouse pre-implantation embryos, compaction occurs at the 8-cell stage around E2.5. During compaction blastomeres flatten against each other and E-cadherin mediated cell–cell adhesion is established. E-cadherin interacts with β-catenin basolaterally [35], stabilised by the Rho GTPases Cdc42 and Rac1 [36], and triggers the apical restriction of microvilli and localisation of a number of proteins including aPKC and Par3 to the apical pole [2]. Recently a Rho GTPase binding protein (Borg5) has been shown to interact with aPKC and Cdc42 and to facilitate the proper apical localisation of aPKC as well as blastocyst formation [37], thus furthering earlier insights into the involvement of Rho GTPases in the establishment of blastomere polarity [38]. In addition, maternal oocyte-derived Cdx2 transcripts contribute to the establishment of cell polarity and embryo development in a mutually reinforcing relationship between Cdx2 and aPKC distribution [39], [40].

Important new insights have recently demonstrated a role of the Hippo signalling pathway in this process. This signalling pathway was first described in Drosophila where it is involved in the regulation of organ size. Its mammalian orthologues have been implicated in biological processes such as epithelial-to-mesenchymal transition and oncogenesis [41]. Crucially, Hippo signalling links cell–cell contact with the transcriptional activity of Tead4 and thereby translates cell positional information into distinct transcriptional outputs and ultimately lineage identity (Fig. 2) [11].

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  • Fig. 2 

    Signalling pathways in pre-implantation embryos determine cell fate. Differential activity of the Ras and Hippo signalling pathways determines lineage segregation in the pre-implantation embryo. Circumferential cell–cell contacts activate the Hippo pathway in the cells located in the interior of the embryo. The transcriptional co-activator Yap becomes phosphorylated and is sequestered in the cytoplasm. Conversely, in exterior cells the Hippo pathway is not activated. Yap is free to translocate to the nucleus where it transactivates Tead4-mediated transcription of key trophoblast transcription factors directly or indirectly [9], [10], [11], [41]. In presumptive embryonic cells, Nanog and Oct4 maintain repression of the trophoblast transcriptional programme in conjunction with epigenetic modifiers like Eset [32], [33], [34], and Elf5 is methylated and repressed [21]. In presumptive trophectoderm cells Cdx2 is activated by Tead4/Yap and in turn induces a self-reinforcing trophoblast-defining transcriptional circuit. Ras–Mapk signalling in the outside cells is also crucial for trophoblast specification and/or integrity [43] but the precise mechanism has yet to be elucidated.

In detail, activation of Hippo signalling in inside cells leads to phosphorylation of the transcriptional co-activators Yap and/or its homologue Taz, which sequesters them to the cytoplasm. By contrast, the pathway remains inactive in outside cells allowing unphosphorylated Yap to translocate to the nucleus where it co-activates the Tead4-mediated cascade of TE specific transcription (Fig. 2) [11], [41]. It has also been shown that it is the circumferential cell–cell contacts of the inside cells that activate the pathway, a finding that correlates well with the involvement of Hippo signalling in growth repression mediated by contact inhibition. The upstream mediators of the Hippo signalling pathway have not been studied as yet in the pre-implantation embryo. It will be interesting to determine if the canonical Hippo activating cell adhesion molecules Fat and Dachsous [42] are also involved in TE/ICM lineage segregation or if alternative mechanisms activate the pathway in the inside cells of the pre-implantation embryo.

Another signalling pathway involved in TE specification is the Ras–Mapk kinase cascade. When this pathway is activated in ES cells by means of a constitutively active H-Ras transgene, transdifferentiation into self-renewing TS cells is observed [43]. This finding provides a rationale for the practice of supplementing ES cell medium with Mapk inhibitors in particular during the derivation of new ES cell lines [44]. The involvement of Ras–Mapk signalling in ES-to-TS cell transdifferentiation is consistent with the requirement of Fgf4 in the derivation and maintenance of TS cells in culture [4]. Like the Hippo signalling pathway it appears that Ras signalling plays a role in TE specification by relaying positional information. Thus, at the 8-cell stage, Mapk1 (Erk2) is localised to the apical edge of the outside blastomeres; when Erk and phospho-Erk levels are reduced through the application of Mapk inhibitors, Cdx2 expression is attenuated and blastocyst formation impaired. Interestingly, genetic ablation of Fgf4 and Fgfr2 as well as exogenous Fgf inhibition does not disrupt initial TE formation [45], [46], [47]. Hence it is likely that Ras–Mapk–Erk activation shifts from an Fgf-independent pathway in pre-implantation development to an Fgf4-dependence for the maintenance of the TS cell compartment in the ExE and in vitro.

With their overlapping function in setting up the lineage-determining transcriptional networks in early development, it is tempting to speculate that the Ras and Hippo signalling pathways intersect in the establishment of the trophoblast lineage. It has been widely documented that Ras induced apoptosis utilises the Hippo signalling pathway through a family of Ras effector proteins (Rassfs) [48], [49], [50]. It will be interesting to determine whether this interaction exists in TE/TS cells.

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6. Can we relate insights from the mouse towards the derivation of human trophoblast stem cells? 

Human trophoblast stem (hTS) cells would constitute a most valuable tool to study genetic and epigenetic control mechanisms of trophoblast proliferation, differentiation and function in normal as well as pathological placentation. This would be particularly advantageous to delineate the earliest developmental processes in a time window when human tissue material is not available. Unfortunately, the derivation of such hTS cells from human blastocysts using the same methods as those established for the mouse has not been successful to date. Surprisingly however, when human ES (hES) cells were derived, some spontaneous differentiation into “trophoblast”-like cells was observed based on the expression of β-HCG, which is in stark contrast to the situation in the mouse where ES cells are largely excluded from differentiating into trophoblast derivatives [51]. Subsequently it was found that this differentiation pathway can be further enhanced by treating hES cells with BMP4, and microarrays confirmed the up-regulation of genes associated with trophoblast function [52]. While these culturing methods favoured differentiation of post-mitotic syncytiotrophoblast cells, stable cytotrophoblast cell lines were successfully derived by repeated β-HCG selection, although the proliferative capacity of these cell lines remained overall low [53]. Recent studies have shown, however, that these ‘trophoblast’-like cells and cytotrophoblast cell lines only recapitulate a partial trophoblast transcriptional programme but do not reflect early placental trophoblast. Thus, in contrast to the situation in the early human placenta where ELF5 is unmethylated and expressed, ELF5 remains fully methylated and repressed in hES-derived trophoblast cells, suggesting that the epigenetic restriction set by ELF5 promoter methylation is not overcome in these cells [54].

Thus, the lineage restricting function of Elf5/ELF5 promoter methylation appears to be conserved between mouse and human. This finding raises the question of whether we can exploit other recent insights into the transcriptional networks, and their regulation, in murine TS cells towards the identification and isolation of an hTS cell population (Table 1). Indeed, a small population of cells has been identified within the villous cytotrophoblast layer of the first trimester human placenta that co-expresses ELF5 and CDX2 and may possess true stem cell characteristics [54]. Additionally, chromatin immunoprecipitation experiments confirmed that the transcriptional circuit between CDX2, EOMES and ELF5 is also conserved in human trophoblast. Thus, criteria that define hTS cell identity likely include ELF5 expression and promoter hypomethylation, the presence of other core transcription factors, namely CDX2 and EOMES, as well as expression of FGFR2 [54], [55]. These criteria should aid the identification of true hTS cell lines in the future.

Table 1. Conserved features in early cell lineage specification and trophoblast differentiation between mouse and human.
Pre-implantation developmentRefs.
Localisation of lineage-determining transcription factorsICM: NANOG, OCT4, DPPA5, REX1, SOX2[57], [58], [59]
TE: CDX2
Epigenetic regulation: DNA methylationTE globally hypomethylated compared to ICM[60], [61]
MAPK signalling pathway componentsTE-enriched[58]

Trophoblast lineage establishment
Trophoblast-reinforcing transcriptional networksMutual activation of CDX2, EOMES and ELF5[21], [54]
Transcriptional regulation of further trophoblast differentiationGATA2/3 and TCFAP2A/C[15], [16], [23], [24], [69], [70]
Epigenetic lineage barrierELF5 hypomethylated and expressed in first trimester villous cytotrophoblast, but DNA methylated and repressed in embryonic lineage derivatives (hES cells)[54]

On further examination, the similarities between human and mouse with respect to the regulation of the trophoblast compartment are even more extensive (Table 1). Although some timing differences may exist, the expression of distinct lineage markers such as CDX2, OCT4 and NANOG is conserved with CDX2 being expressed in the TE and NANOG and OCT4 demarcating the ICM of human blastocysts [56], [57], [58], [59]. The TE of human blastocysts is also particularly enriched in expression of RAS–MAPK pathway components, emphasizing the importance of this signalling cascade for trophoblast development [58]. The similarities in early cell lineage specification extend to the epigenetic landscape with a global hypomethylation of the TE compared to the ICM that is observed in both mouse and human blastocysts [60], [61]. Further, in human as in mouse ES cells, the pluripotency factors OCT4, NANOG and SOX2 appear to have a role in repressing the trophoblast transcriptional programme [62], [63], [64], [65], [66], [67].

The similarities are not limited to initial lineage commitment. Human GATA2 and GATA3 are expressed in the placenta and, like mouse Gata3, have been found to play a role in the further differentiation of trophoblast, driving trophoblast (stem) cell differentiation into syncytiotrophoblast [68], [69]. In addition, TCFAP2A and TCFAP2C are both detected in the human placenta and their expression may affect trophoblast differentiation and invasion [70]. Thus it seems likely that much of the trophoblast-defining transcriptional programme, and possibly its regulation, is conserved between mice and men.

However, it may also be possible that despite the similarities of the transcriptional networks and signalling pathways, some culture requirements for TS cell derivation and maintenance may differ between human and mouse. This may go some way to explaining the lack of success in deriving hTS cells from human blastocysts using culture conditions required for derivation of mTS cells. Interestingly, the derivation of vole TS cell lines was recently reported without the need for Fgf4 in the culture medium [71]. Given the divergent requirements of two rodent species it is feasible that hTS cells may require supplementation with some different or additional cytokines and/or growth factors to maintain the TS transcriptional programme and consequently TS cell self-renewal.

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7. Conclusion 

Recent insights from studies in the mouse have shed light onto the molecular pathways that translate cell–cell contact information into transcriptional programmes to define the trophoblast cell lineage. These programmes are reinforced by epigenetic mechanisms acting on different levels to ensure stable trophoblast cell fate. An increasingly detailed knowledge of transcription factor networks in trophoblast stem cells has highlighted the lability of the stem cell state, which may have evolved to prevent the persistence of trophoblast cells with stem cell potential in the maternal uterus and may underlie the notorious difficulty to maintain the proliferative capacity of trophoblast cell lines. Despite the anatomical differences in the placentas of different species, it seems that the molecular mechanisms of early trophoblast specification and establishment of a proliferative stem cell-like compartment are largely conserved between mice and humans. Thus it may be possible that we can learn from our insights in the mouse to better understand early trophoblast differentiation in the human conceptus, both in healthy and pathological pregnancies. These advances may enable us to identify stem cell compartments in the early human placenta and to translate the requirements for stem cell self-renewal for the successful derivation hTS cell lines truly representative of first trimester trophoblast.

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Acknowledgements 

Work in the Hemberger lab is supported by the Biotechnology and Biological Sciences Research Council, UK.

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PII: S0143-4004(10)00282-1

doi:10.1016/j.placenta.2010.07.013

Placenta
Volume 31, Issue 11 , Pages 944-950, November 2010

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