Directed conversion of porcine expanded pluripotent stem cells into trophoblast stem cells.

Trophoblast stem cells (TSCs) serve as a valuable system for investigating the signaling pathways that regulate extraembryonic lineage commitment. Although trophoblast specification has been extensively characterized in humans and rodents, comparable tools and molecular frameworks remain underdeveloped in pigs, a species of significant agricultural and biomedical relevance. This study identifies defined biochemical conditions that enable efficient conversion of porcine (p) expanded pluripotent stem cells (EPSCs) into induced TSCs (iTSCs). The results demonstrate that combined inhibition of TGF-β/Activin and MEK/ERK signaling is sufficient to induce and maintain core trophoblast regulators, including CDX2, GATA3, KRT7/18, HAND1, and ELF5, while suppressing pluripotency networks. Transcriptomic analyses, benchmarked against porcine in vivo trophoblast reference datasets, indicate that piTSCs closely resemble peri-implantation and term trophoblast populations, confirming successful lineage conversion from the pluripotent state. In vivo, piTSCs formed only small, lineage-restricted lesions lacking ectodermal and endodermal derivatives and exhibiting widespread immunoreactivity for trophoblast markers. In contrast, parental pEPSCs generated teratomas containing derivatives of all three germ layers. Collectively, these findings establish a robust experimental framework for trophoblast induction and characterization in pigs, providing a model to investigate conserved and species-specific mechanisms of trophoblast development and placental biology.