Reconstructing the emergence of the human chorion via HIPPO-mediated trophoblast induction

Abstract

The first lineage decision in the mammalian blastocyst commits outer cells to the trophectoderm and initiates the trajectory that gives rise to the placental chorion. The molecular sequence that unfolds downstream of HIPPO pathway inactivation, linking human trophectoderm specification to the early organization of the chorion, has remained unknown. Here, we establish a developmentally informed model that leverages HIPPO pathway modulation to induce the native trophectoderm trajectory in the absence of exogenous BMP or WNT signaling. We first transiently reset primed human pluripotent stem cells into a trophectoderm-competent ground state, followed by LATS kinase inhibition to set the trajectory in motion. To benchmark fidelity, we built an embryo-chorion single-cell reference integrating published early human and placental transcriptomes and applied a computational stage-matching tool to align our cultures to natural development. Stage matching revealed an ordered progression along the trophectoderm trajectory from early TE to post-implantation trophoblast. With extended culture, all major cell types of the nascent chorion emerged, encompassing both trophoblast and chorionic mesoderm lineages. Within the trophoblast, we identified proliferative and non-cycling villous cytotrophoblast, a columnar population connecting villous and extravillous domains, as well as syncytia and extravillous subtypes. When cultured in suspension, these lineages self-organized into three-dimensional organoids that recapitulated the stromal-epithelial architecture and proliferative-syncytial polarity of the emergent chorion. We identified CLDN6 as a defining surface marker of columnar trophoblast, the population that bridges villous and extravillous compartments. Prospective isolation of living CLDN6+ trophoblast revealed their capacity to reacquire a proliferative villous state and, under directed cues, generate both syncytial and extravillous fates, confirming their proposed dual developmental potential within the chorion. Together, these findings establish a developmentally informed framework that connects human trophectoderm specification to the emergent chorion and provides a dynamic platform for investigating the earliest steps of placental specification and the origins of implantation disorders.