Conversion from mouse embryonic to extra-embryonic endoderm stem cells reveals distinct differentiation capacities of pluripotent stem cell states

Abstract

SUMMARYThe inner cell mass of the mouse pre-implantation blastocyst comprises epiblast progenitor and primitive endoderm cells of which cognate embryonic (mESCs) or extra-embryonic (XEN) stem cell lines can be derived. Importantly, each stem cell type retains the defining properties and lineage restriction of their in vivo tissue of origin. Recently, we demonstrated that XEN-like cells arise within mESC cultures. This raises the possibility that mESCs can generate self-renewing XEN cells without the requirement for gene manipulation. We have developed a novel approach to convert mESCs to XEN cells (cXEN) using growth factors. We confirm that the downregulation of the pluripotency transcription factor Nanog and the expression of primitive endoderm-associated genes Gata6, Gata4, Sox17 and Pdgfra are necessary for cXEN cell derivation. This approach highlights an important function for Fgf4 in cXEN cell derivation. Paracrine FGF signalling compensates for the loss of endogenous Fgf4, which is necessary to exit mESC selfrenewal, but not for XEN cell maintenance. Our cXEN protocol also reveals that distinct pluripotent stem cells respond uniquely to differentiation promoting signals. cXEN cells can be derived from mESCs cultured with Erk and Gsk3 inhibitors (2i), and LIF, similar to conventional mESCs. However, we find that epiblast stem cells (EpiSCs) derived from the post-implantation embryo are refractory to cXEN cell establishment, consistent with the hypothesis that EpiSCs represent a pluripotent state distinct from mESCs. In all, these findings suggest that the potential of mESCs includes the capacity to give rise to both extra-embryonic and embryonic lineages. KEY WORDS: Pluripotent stem cells, Directed differentiation, Extra-embryonic endoderm, FGF, Mouse embryoConversion from mouse embryonic to extra-embryonic endoderm stem cells reveals distinct differentiation capacities of pluripotent stem cell states RESEARCH ARTICLE Conversion of mES to cXEN cellsNevertheless, it remains unclear whether self-renewing XEN cells can be derived directly from mESCs without requiring transgenic over-expression.The fibroblast growth factor (FGF) receptor Fgfr2 is enriched in PrE cells, and the ligand Fgf4 is expressed by epiblast progenitor cells within the ICM (Feldman et al., 1995;Arman et al., 1998; Guo et al., 2010). This complementary receptor-ligand expression suggests that epiblast-secreted Fgf4 may be functionally important for PrE development (Rappolee et al., 1994; Goldin and Papaioannou, 2003). It has recently been suggested that PrE formation requires non-cell-autonomous provision of Fgf4 by Nanog-expressing cells (Nichols et al., 2009;Messerschmidt and Kemler, 2010;Yamanaka et al., 2010; Frankenberg et al., 2011). Indeed, Fgf4 or Fgf2, which are not expressed in the early embryo, both function via Fgfr2 and are routinely added during XEN derivation from embryos (Kunath et al., 2005). However, it is unclear whether FGFs are required for XEN cell derivation or whether they function in an autocrine or paracrine m...

Cdx2Efficiently Induces Trophoblast Stem-Like Cells in Naïve, but Not Primed, Pluripotent Stem Cells

“…Our proposal that primed pluripotent cells resist forming TSC-like cells is consistent with prior observations that EpiSCs are resistant to forming XEN cells, while ESCs and iPSCs give rise to XEN cells readily [12]. Our model is also consistent with prior observations that EpiSCs are resistant to forming trophoblast following growth factor treatment [53].…”

“…In addition, both human and mouse ESCs and iPSCs can exist in either of two pluripotent states, termed ground state and naïve pluripotency [9][10][11]. Recent studies have begun to investigate whether differences in the pluripotent state influence each cell line's ability to reproducibly differentiate into specific lineages during directed in vitro differentiation [9,12,13]. Resolving the differences in in vitro differentiation among these cell types will critically inform the decision as to whether new stem cell models are equivalent to or can effectively replace ESCs as both a model for basic biology and as a tool for regenerative medicine.…”

“…Similarly, TSCs can be converted to ESC-like iPSC by overexpressing Oct4 [24,25]. Likewise, ESCs can be converted to XEN cells using growth factors or PE transcription factors [12,[26][27][28][29]. Interestingly, differences in the pluripotent state influence the ability of pluripotent stem cell lines to give rise to XEN cell lines [12].…”

“…Likewise, ESCs can be converted to XEN cells using growth factors or PE transcription factors [12,[26][27][28][29]. Interestingly, differences in the pluripotent state influence the ability of pluripotent stem cell lines to give rise to XEN cell lines [12]. Whether CDX2 efficiently induces formation of TSC-like cells in EpiSCs or ECCs has not been examined, but would provide new insight into the developmental potential of the various pluripotent stem cell states.…”

The XEN of reprogramming

“…Therefore, somatic cells in the adult are not even its developmental descents. However, although the developmental fates of epiblast and XEN are divergent from each other in vivo , XEN cells have been derived from PSCs in vitro [12]. These observations combined with the observation of XEN-to-PSC transition by Zhao et al [5] indicate that cells can be more plastic in vitro, and the chemical cocktail of Deng's group might be hijacking such plasticity of XEN cells instead of reversing ontogeny.…”