Transient inhibition of MEK/ERK and WNT pathways enhances direct differentiation of primed hPSCs into functional trophoblast stem cells

Abstract The placenta plays a pivotal role in human pregnancy, yet research into placental development has been hindered by limited access to early-stage embryos and ethical constraints. Although human trophoblast stem cells (hTSCs) have been established from blastocysts, deriving these cells efficiently from primed human pluripotent stem cells (hPSCs) remains challenging. Here, we developed a simplified and efficient strategy that enables direct, efficient conversion of primed hPSCs into stable, self-renewing hTSCs by transiently inhibiting the MEK/ERK signaling pathway using the inhibitor PD0325901 in a simplified basal medium. This approach significantly enhanced the generation of trophoblast cells expressing the critical trophoblast marker GATA3 and led to the establishment of homogeneous hTSC lines with robust capacities to differentiate into functional extravillous trophoblast (EVT) and syncytiotrophoblast (STB) lineages. Transcriptomic and chromatin accessibility analyses confirmed that these hTSCs closely resembled blastocyst-derived trophoblast cells and clearly differed from amnion lineages, confirming authentic trophoblast identity distinct from amnion. Additionally, precise modulation of WNT signaling activity was essential for optimal trophoblast induction efficiency, highlighting the importance of signaling equilibrium in trophoblast differentiation. Collectively, our optimized protocol offers an accessible and reproducible platform for modeling early placental development and understanding the pathogenesis of trophoblast-associated disorders in vitro.