Traditionally ESCs were derived and cultured in the presence

Traditionally, ESCs were derived and cultured in the presence of leukemia inhibitory factor (LIF) and either bone morphogenetic protein 4 (BMP4) or fetal bovine serum (BMP4/L or FBS/L) (Ying et al., 2003a). Under such conditions, ESC cultures are heterogeneous and contain metastable and fluctuating subpopulations, resembling later (post-implantation epiblast) or earlier (two-cell stage) developmental stages (Hayashi et al., 2008; Macfarlan et al., 2012). Recently, efficient and clonal derivation from ICM cells (Boroviak et al., 2014) was reported by using a defined medium containing two inhibitors of MEK and GSK3β kinases together with LIF (2i/L). ESC lines cultured in 2i/L maintain a less heterogeneous “naive” ground state (Marks et al., 2012; Ying et al., 2008). Early in development, PDGFRα has a relatively weak but well visible expression in all blastomeres until it becomes stronger in PrE-committed cells around E3.75 (around 64 cells) (Artus et al., 2011; Grabarek et al., 2012; Plusa et al., 2008). Here, we demonstrate that PDGFRα+ cells can also be identified in undifferentiated ESC cultures. The PDGFRα+ subpopulations show a unique PrE-primed molecular and epigenetic signature, which is reflected by functional in vitro and in vivo differences when compared with the epiblast counterpart (PECAM1+). Despite these differences, the transcriptome of PDGFRα+ cells displays similarities with naive ESCs and with early/mid scopolamine hydrobromide cells. These findings suggest that PDGFRα+ cells are the equivalent of the in vivo PrE (hypoblast) precursors present at the pre-implantation stage.
Results
Discussion During development, PDGFRα has an early, relatively weak but well visible expression from morula stage onward until it becomes stronger in PrE-fated cells at ∼E3.75 (64 cells) (Artus et al., 2011; Grabarek et al., 2012; Plusa et al., 2008). In this study, we investigated its presence in undifferentiated ESCs, further dissecting their known heterogeneity. By taking advantage of the endogenous expression of PECAM1 and PDGFRα, we defined three different subpopulations that were further characterized (Figures 1 and 2): PECAM1+/PDGFRα− (epiblast-primed), PECAM1+/PDGFRα+ (double-positive) and PECAM1-/PDGFRα+ (PrE-primed) cells. PrE-primed cells have a distinct molecular identity, as they co-express OCT4, GATA4, GATA6, and SOX17, which differs from epiblast-primed cells, which co-express OCT4, NANOG, and SOX2. Double-positive cells appear to be an intermediate between epiblast- and PrE-primed cells. In line with this, we also identified, at the single-cell level, cells co-expressing OCT4, GATA4, and NANOG. This is reminiscent of the simultaneous expression of epiblast and extraembryonic determinants in early pre-implantation development (Guo et al., 2010). Although PrE-biased cells have already been described as Hex (Canham et al., 2010; Morgani et al., 2013), PDGFRα+ cells have a more pronounced PrE phenotype and a lower expression of epiblast determinants, which are still retained in Hex cells (Figure S5). Moreover, our model does not rely on signal amplification and on the use of reporter lines (Canham et al., 2010; Morgani et al., 2013), allowing the separation of these subpopulations in every ESC line of interest. In vitro features of the three subpopulations appear to be drastically divergent in terms of self-renewal and differentiation capacity (Figure 3). Epiblast-primed cells and double-positive cells but not PrE-primed cells could re-establish the initial heterogeneity. When addressing their differentiation potential, PrE-primed cells efficiently generated XEN-like cells but not embryonic or presumptive trophoblast types diversely from epiblast-primed subpopulation. PDGFRα+ cells have a distinct epigenetic state, characterized by a lower level of DNA methylation/hydroxymethylation and by a different pattern of epigenetic regulators (Figure 4), in line with the notion that extraembryonic tissues (Sakaue et al., 2010) and their stem cell models (Rugg-Gunn et al., 2010) are hypomethylated.