Directed conversion of porcine extended pluripotent stem cells into trophoblast-like stem cells through modulation of conserved TGF-β and ERK signaling pathways

Trophoblast stem cells (TSCs) provide a tractable system for interrogating the signaling pathways that govern extraembryonic lineage commitment. Although trophoblast specification has been extensively characterized in humans and rodents, comparable tools and molecular frameworks remain poorly defined in pigs. Here, we identify defined biochemical conditions that enable the conversion of porcine extended pluripotent stem cells (pEPSCs) into TSCs. Pharmacological inhibition experiments demonstrate that coordinated repression of TGF-{beta}/Activin and MEK/ERK signaling is sufficient to induce and maintain a stable trophoblast transcriptional program. Under these conditions, cells robustly upregulate core trophoblast regulators, including CDX2, GATA3, KRT7/18, HAND1, and ELF5, while concomitantly suppressing pluripotency- and hypoblast-associated gene networks. Bulk transcriptomic profiling reveals extensive lineage reprogramming, with enrichment of pathways related to cell adhesion, extracellular matrix organization, and placental development. Functional in vivo assays further show that induced trophoblast-like cells form small, non-teratomatous lesions that express extraembryonic markers, whereas parental pEPSCs generate teratomas that contain derivatives of all three germ layers. Together, these findings establish that combined inhibition of TGF-{beta}/Activin and MEK/ERK signaling is sufficient to specify porcine trophoblast identity from pluripotent stem cells and provide a biochemical framework for dissecting conserved and species-specific mechanisms underlying trophoblast specification and placental development. HighlightsO_LIDefined signaling conditions enabling stable conversion of porcine extended pluripotent stem cells (EPSCs) into trophoblast-like stem cells (TSC). C_LIO_LIDual inhibition of TGF-{beta}/Activin and ERK pathways drives robust trophoblast commitment. C_LIO_LITranscriptional reprogramming reveals conserved trophectoderm regulatory networks distinct from pluripotency and hypoblast states. C_LIO_LIInduced porcine TSCs display restricted in vivo potential, consistent with trophoblast identity. C_LI