Building Efficient Stem Cell-Derived Mouse Blastocyst Models for Environmental Studies

Stem cell-based embryo models offer a powerful platform for investigating early mammalian development and its sensitivity to environmental perturbations. Here, a robust and accessible protocol is presented for generating high-fidelity mouse blastoids using embryonic stem cells (ESCs), trophoblast stem cells (TSCs), and embryonic stem cells engineered for inducible GATA4 expression (iGATA4-ESCs). This system produces iGATA4-blastoids that accurately recapitulate natural E4.5 blastocyst morphology, lineage proportions, and transcriptional signatures, providing a reliable in vitro model of the primitive endoderm (PE), epiblast (EPI), and trophectoderm (TE) compartments. This protocol first demonstrates how to maintain and prepare the three stem cell populations required for blastoid formation. The step-by-step assembly of iGATA4-blastoids under pre-implantation culture conditions is then outlined, highlighting critical experimental parameters. To enable rigorous characterization, a tested workflow for blastoid fixation, staining, and imaging is provided. Finally, an example is presented demonstrating how iGATA4-blastoids can be used for high-throughput assays. By exposing large cohorts of blastoids to defined environmental factors, changes in morphology, lineage allocation, and developmental progression can be quantified rapidly and reproducibly. Together, this protocol establishes a scalable platform for modeling pre-implantation development, dissecting early lineage decisions, and performing multiparametric screens to assess environmental or molecular influences on blastocyst formation.