Signaling Induced Plasticity Enables Transgene-Free Generation of Mouse Post-Gastrulation Whole Embryo Models Solely from Naïve ESCs and iPSCs (i)

The ability to generate synthetic mouse embryo models (SEMs) that can grow beyond gastrulation and early organogenesis has proven the plasticity of naïve Pluripotent stem cells (nPSC) to fully recapitulate the developmental dynamics of the mouse embryo. Existing protocols rely on the induction of extraembryonic lineages by ectopically expressing master transcription factors that induce their differentiation to trophoblast (TE) or primitive endoderm (PrE) that latter will form the placenta and yolk sac, respectively. However, this approach brings technical difficulties related to the required genetic manipulation, separate lineage induction and mixing of different cell lines. While several works have suggested efficient priming to extraembryonic lineages without the need of transgenes, they have not proven the capability of this cells to contribute to the formation of SEMs. In this study, we demonstrate that naive ESCs can be co-induced in a single transgene free condition to give rise to both embryonic and extraembryonic lineages present in the preimplantation mouse embryo while preserving an epiblast compartment. Furthermore, this condition can be aggregated and cultured, resembling post-implantation egg cylinders, and by applying ex-utero growth technologies they progress to mimic features corresponding to E8.5 in-utero growth embryos. Moreover, by optimization of a media termed Alternative Condition (AC), we provide ready to use condition that can be maintained long term and directly aggregated to generate TF-SEMs, eliminating the need of cell preinductions before aggregation.