Efficient mouse embryo models from stem cells for environmental studies

Blastoids are stem cell-derived structures that mimic natural blastocysts, including formation of the trophectoderm, epiblast, and primitive endoderm lineages. However, current methods often produce incomplete structures that do not cavitate or form sufficient primitive endoderm. We developed a new approach to enhance blastoid formation by aggregating embryonic stem cells (ESCs) with ESCs transiently expressing Gata4 (iG4-ESCs) to promote the primitive endoderm fate, followed by addition of trophoblast stem cells (TSCs). This method achieved an ~80% efficiency in forming cavitated blastocyst-like structures, termed iG4-blastoids. Single-cell RNA sequencing revealed that iG4-blastoids resemble day 4.5 blastocysts. iG4-blastoids cultured in media lacking FGF4 showed improved specification of the invasive mural trophectoderm. iG4-blastoids underwent post-implantation remodeling, with ~12% forming an egg cylinder-like structure. Notably, blastoid formation was affected by caffeine, alcohol, nicotine, and amino acid levels. Thus, iG4-blastoids are a robust model for screening diverse environmental factors for their consequences on embryogenesis.