Jeoung, Y.-H. | Search bioRxiv

bioRxiv2026

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

developmental biology↗

bioRxiv2025

A porcine germ cell depletion model to investigate the role of germ cells in gonadal development.

The molecular mechanisms regulating the germ cell and gonadal somatic cell development are complex and are still poorly understood in non-rodent mammals. The Nanos family gene-NANOS3 is expressed in the migrating primordial germ cells (PGC) and has key roles in germ cell survival in several species. Herein, we investigated the role of NANOS3 in pigs-an animal model of dual agricultural and biomedical importance. Consistent with prior reports, we found that disruption of NANOS3 expression did not affect PGC specification. Similarly, migration of PGC to the genital ridge occurs normally, but soon followed by an increase in apoptosis and a loss of undifferentiated state by embryonic (e) day 20. Although NANOS3-/- gonads initiate development as ovaries or testes, they displayed structural defects. This is especially pronounced in the ovaries after the sex determination period (e35). Notably, we identified defects in pregranulosa and theca cell development at a single-cell resolution, suggesting that germ cells are important for gonadal somatic cell development. This germ cell depletion model therefore offers novel insights into the potential role of germ cells in the emergence of gonadal somatic lineages.

developmental biology↗