A single cell trajectory of human archetypal pluripotent stem cell differentiation to trophoblast stem cells reveals induction of endogenous BMP5/7 and GATA3 without transitioning through a naive state

The human placenta is increasingly a focus of research related to early child development and the impact of maternal hyperimmune states. Primary human trophoblast stem cells (hTSC) and human pluripotent stem cells (hPSC) differentiated to hTSC can potentially model placental processesin vitro. Yet, it remains controversial how the differentiation of human pluripotent stem cells to trophectoderm relates toin vivodevelopment and the factors required for this differentiation. Here, we demonstrate that the primed pluripotent state retains potency to generate trophoblast stem cells by activating EGF and WNT and inhibiting TGFb, HDAC and ROCK signaling without exogenous BMP4 (named TS). We map this specification by temporal single cell RNAseq compared to activating BMP4 or activating BMP4 and inhibiting WNT. TS conditions generate a stable proliferating cell type that is highly similar to six-week placental cytotrophoblasts with activation of endogenous retroviral genes and without amnion expression. Multiple primed iPSC and ES lines differentiate to iPS-derived-TSCs that can be passaged for at least 30 passages and differentiate to pure populations of multinucleated syncytiotrophoblasts and extravillous trophoblast cells. Our findings establish that primed iPS cell specification to hTSC with TS conditions involves induction ofTMSB4X,BMP5/7, GATA3 andTFAP2Awithout transitioning through a naive state. Collectively, our results suggest that the primed state is on a continuum of potency and can differentiate to trophoblast stem cells via multiple paths.