Failure of achieving proper segregation resulted from disproportionate and aberrant co-assembly between the three cell types following initial aggregation ( Figure S2A).
Correct segregation and localization of each cell type was observed in approximately 25% of the sEmbryos ( Figure 1G). Immunostaining for Oct4 (epiblast ), Tfap2c (E圎), and Sox17 (VE) at day 4 corroborated proper expression of lineage markers by each donor cell population ( Figure 1H). WT-blue flourescent protein (BFP)-labeled ESCs predominantly contributed to the embryo proper, and iGata4-GFP-labeled cells localized to the visceral endoderm (VE) surrounding the sEmbryos ( Figure 1F–H). iCdx2-mCherry lines correctly localized to the extraembryonic ectoderm (E圎) compartment in egg-cylinder-shaped embryos ( Figure 1F–H). Each of the lines was transduced with a lentivirus constitutively expressing a different fluorescent label ( Figure 1E). We tested whether assembling the three naive ESCs lines (WT, iGata4, and iCdx2) following DOX pre-treatment and co-aggregation up to day 5 can result in egg-cylinder like structures in vitro and whether the cells segregate based on their transgenic priming ( Figure 1D). Our findings highlight the plastic potential of naive pluripotent cells to self-organize and functionally reconstitute and model the entire mammalian embryo beyond gastrulation. sEmbryos adequately accomplish gastrulation, advance through key developmental milestones, and develop organ progenitors within complex extraembryonic compartments similar to E8.5 stage mouse embryos. This was achieved by co-aggregating non-transduced ESCs, with naive ESCs transiently expressing Cdx2 or Gata4 to promote their priming toward trophectoderm and primitive endoderm lineages, respectively. Here, we adapt a recently established platform for prolonged ex utero growth of natural embryos to generate mouse post-gastrulation synthetic whole embryo models (sEmbryos), with both embryonic and extraembryonic compartments, starting solely from naive ESCs. However, whether cultured stem cells can independently give rise to entire gastrulating embryo-like structures with embryonic and extraembryonic compartments remains unknown. In vitro cultured stem cells with distinct developmental capacities can contribute to embryonic or extraembryonic tissues after microinjection into pre-implantation mammalian embryos.
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