Ex Utero Development of Post-Gastrulation Synthetic Mouse Embryos Generated Solely from Naïve Pluripotent Stem Cells [RNA-seq]

Different types of in vitro expanded stem cells can contribute to embryonic or extra-embryonic compartments after microinjection into blastocysts. However, whether stem cells could give rise to advanced gastrulating whole embryo-like structures with both embryonic and extra-embryonic compartments, without relying on a micro-injected host embryo, remains to be achieved. Thus far, in vitro aggregated stem cells have failed to generate post-gastrulation embryos ex utero or in utero, which partially resulted from the lack of methods for prolonged expansion of embryos until advanced developmental stages ex utero. Here we adapt recently optimized platform and conditions for ex utero growth of natural embryos, to generate complete mouse synthetic embryos, with both embryonic and extra-embryonic compartments, and by starting solely from naïve ESCs. The latter is achieved following co-aggregating non-transduced naïve ESCs, with Cdx2- and Gata4- transiently pulsed naïve ESCs, to promote their trophoblast and primitive endoderm lineage induction, respectively. The obtained synthetic embryos adequately advance in this platform through developmental milestones and accomplish gastrulation and develop organs progenitors within normally developed extra-embryonic compartments. Our findings highlight the plastic developmental potential of the naïve pluripotent state to functionally self-organize and reconstitute the entire early mammalian embryo. In this study, we induced mouse embryonic stem cells (ESC) into trophoblast stem cell (TSC) fate. We used the KH2 collagen flip-in ESC system that carriers M2RtTa allele in the Rosa26 locus, and flipped-in Cdx2 into the collagen locus under the regulation of Doxycycline (DOX) inducible Tet-On promoter. These iCdx2 ESCs were treated with Doxycyclne in different durations, with and without LPA, a Hippo pathway inhibitor that induces YAP nuclear localization. In addition to iCdx2 ESCs, we used established TSCs, WT ESCs, MEF (mouse embryonic fibroblasts) and XEN (mouse extra-embryonic endoderm) cells as control.