Single-cell analysis of human trophoblast stem cell specification reveals activation of fetal cytotrophoblast expression programs including coronavirus associated host factors and human endogenous retroviruses

The human placenta is increasingly a focus of research related to early child development and the impact of maternal hyperimmune states. The ability to model human trophectoderm disease states from human pluripotent stem cells, the nature of human pluripotent stem cell potency and the mechanisms regulating human trophectoderm specification remains poorly understood. Recent work suggests that only the naive state can give rise to trophectoderm and that primed iPSC generate mixed amnionic and mesoderm lineages. Here we identify conditions that efficiently drive the specification of primed iPSC to trophectoderm, named Trophoblast Stem Cell (TSC). iPS-derived-TSC share transcriptional, morphological and functional characteristics with human in vivo cytotrophoblasts including activation of human endogenous retroviruses, expression of COVID-19 associated host factors and generation of multinucleated syncytiotrophoblasts with a large fusion index. At high densities in 5% O2, iPS-derived-TSC form villi-like structures and express extravillous and syncytiotrophoblast proteins HCG-{beta} and HLA-G. Using temporal single cell RNAseq, we define the molecular changes associated with specification under three separate conditions: 1) BMP4, 2) BMP4 and inhibition of WNT, 3) activation of EGF and WNT, inhibition of TGFbeta, HDAC and ROCK signaling (named TSC). With 9,821 high-quality single cell transcriptomes, we find that BMP4 gives rise to mesenchymal cells while TS conditions lacking exogenous BMP4 generate a stable proliferating cell type that is highly similar to six week placenta cytotrophoblasts. TFAP2A programs the specification of primed iPS cells to TSC without transitioning through a naive state. TSC specification independent of exogenous BMP4 will allow for robust and reproducible studies of the cytotrophoblast component of human placenta.