Transcription factor-mediated reprograming is a powerful method to study cell fate changes. In this study, we demonstrate that the transcription factor Gata6 can initiate reprograming of multiple cell types to induced extraembryonic endoderm stem (iXEN) cells. Intriguingly, Gata6 is sufficient to drive iXEN cells from mouse pluripotent cells and differentiated neural cells. Furthermore, GATA6 induction in human embryonic stem (hES) cells also down-regulates pluripotency gene expression and up-regulates extraembryonic endoderm (ExEn) genes, revealing a conserved function in mediating this cell fate switch. Profiling transcriptional changes following Gata6 induction in mES cells reveals step-wise pluripotency factor disengagement, with initial repression of Nanog and Esrrb, then Sox2, and finally Oct4, alongside step-wise activation of ExEn genes. Chromatin immunoprecipitation and subsequent high-throughput sequencing analysis shows Gata6 enrichment near pluripotency and endoderm genes, suggesting that Gata6 functions as both a direct repressor and activator. Together, this demonstrates that Gata6 is a versatile and potent reprograming factor that can act alone to drive a cell fate switch from diverse cell types.

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...

At the time of implantation in the maternal uterus, the mouse blastocyst possesses an inner cell mass comprising two lineages: epiblast (Epi) and primitive endoderm (PrE). Representative stem cells derived from these two cell lineages can be expanded and maintained indefinitely in vitro as either embryonic stem (ES ) or XEN cells, respectively. Here we describe protocols that can be used to establish XEN cell lines. These include the establishment of XEN cells from blastocyst-stage embryos in either standard embryonic or trophoblast stem (TS ) cell culture conditions. We also describe protocols for establishing XEN cells directly from ES cells by either retinoic acid and activin-based conversion or by overexpression of the GATA transcription factor Gata6. XEN cells are a useful model of PrE cells, with which they share gene expression, differentiation potential and lineage restriction. The robust protocols for deriving XEN cells described here can be completed within 2–3 weeks.