The early mammalian embryo (blastocyst) contains three co-developing types of stem cells. Two supporting extraembryonic types -the trophectoderm and the primitive endoderm -encase and guide the pluripotent epiblast that eventually forms all body tissues. Unlike embryos, stem cell-based models of the embryo can be generated in large numbers and subjected to high-content screens as a basis for basic and biomedical discoveries (Rivron et al., 2018a;Vrij et al., 2016b). Here, we show that aggregates of naive Embryonic stem cells cultured in chemically-defined conditions and exposed to combinatorial screens of signaling molecules, rapidly (48 hours) and efficiently (80%) set apart PrE-like cells. These cells produce a basal lamina, generate progenitors resembling both visceral and parietal endoderm, and polarize co-developing Epiblast cells to form a proamniotic cavity. In blastoids, stem cell-based models of the early blastocyst (Rivron et al., 2018b), this combination of signals increases the ratio and number of Gata6+/Nanog+ cells and promotes the survival, growth and morphogenesis of a post-implantation-like Epiblast in vitro. Modeling early embryonic development in chemically-defined in vitro conditions shows that the primitive endoderm forms via a specific combination of signaling pathways and sufficient to drive the development of the Epiblast. Hamazaki et al., 2004). However, EBs are formed in chemically-undefined conditions and PrE is not generated efficiently, which occlude studying underlying mechanisms. Recently, we reported the generation of blastoids (Rivron et al., 2018b), embryonic stem cell (ESC) and trophoblast stem cell (TSC)-based structures resembling the E3.5 blastocyst. Building on previous observations (Gardner, 2000), this model proposed a range of inductive signals originating from the embryo and regulating trophectoderm development and implantation. Here, we investigate the role of signaling pathways forming the PrE under chemically-defined conditions, and its influence on post-implantation development of blastoids.
Results
Naïve pluripotency increase PrE differentiation.We used a high-content screening platform of non-adherent hydrogel microwells in 96 well-plates (Vrij et al., 2016a) to reproducibly aggregate small number of ESCs into EBs (Figure 1A). ESCs seeded into microwells were normally distributed across the 430 microwells within each well (7-12 cells per microwell) and formed EBs within 24 hours (Figure 1B, S1). We quantified PrE differentiation via in situ imaging of fluorescent reporters Pdgfrα (ESCs Pdgfrα-h2b-gfp/+ ) (Artus et al., 2010;Plusa et al., 2008) or Gata6 ESCs Gata6-h2b-venus/+ ). Leukemia inhibitory factor (Lif) appeared essential for the viability of EBs when formed with low cell numbers in serum-free B27N2 medium. However, EBs did not proliferate and formed only few PrE cells (1% and 4% of Pdgfrα+ EBs for 2D expansion in B27N2/2i/Lif and serum/Lif, respectively (Figure 1C). In contrast, serum/Lif induced proliferation and expression of Pdgfrα+ EBs (44%, Figure 1C, ...
