BMP4 and FGF2 promote heterogeneous priming including extraembryonic lineages in porcine expanded potential stem cells.

INTRODUCTION: Extraembryonic lineages, specifically the trophectoderm (TE) and primitive endoderm (PrE), are indispensable for early embryogenesis and successful pregnancy. Expanded potential stem cells (EPSCs) can contribute to both embryonic and extraembryonic fates, providing a platform to model early lineage specification. METHODS: This study investigated the effects of fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 4 (BMP4) (2F condition) on lineage-associated priming in porcine EPSCs (pEPSCs). RESULTS: Following 2F treatment, cells maintained a normal karyotype and underwent morphological changes accompanied by downregulation of pluripotency markers (OCT4, SOX2) and upregulation of TE (CDX2 and GATA3) and PrE markers (GATA4, GATA6, SOX17, and HNF4A). Transcriptomic and immunofluorescence analyses consistently demonstrated activation of TE- and PrE-associated molecular programs, although markers of additional developmental lineages were also detected. These findings suggest that combined FGF2 and BMP4 treatment promotes extraembryonic lineage-associated priming in pEPSCs, and generates a heterogeneous population exhibiting features of both TE- and PrE-associated states. DISCUSSION: Collectively, this study establishes a simple and defined in vitro platform for investigating signaling-dependent lineage priming and cell state remodeling in porcine EPSCs.