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. Overall design: Following iterative tests and optimizations, we formulated the following regimen and media compositions as most optimal to obtain Epi, PrE and TE cells within 4 days from 2i/LIF naïve ESCs (sample A). Our 4 day regimen consists of 24h treatment of Multilineage Promoting Media 1 (MLPM1) which contains N2B27 based media with CHIR – REPSOX – AM580 and NaB (sample B), followed by 24h treatment of MLPM2 which is MLPM1 that lacks NaB, and has CHIR concentration reduced from 6 µM to 3 µM, and in which LIF is reintroduced (sample C). Next, the cells were then harvested and aggregated in AggreWell plates, with 30 cells per microwell as employed on previous transgene mediated SEM protocols, for 2 days in Multilineage Maturation Media (MLMM – N2B27 with FGF4, ACTIVIN A, XAV and TRULI) (sample D). Using the AC media, we maintained PrE, TE and Epi-like cells for 12 passages on mouse embryonic fibroblast layers and harvested these cells for sequencing.