Single-cell transcriptomics reveals differences between chorionic and basal plate cytotrophoblasts and 2D-cultured trophoblast stem cells.

Cytotrophoblast (CTB) of the early gestation human placenta are bipotent progenitor epithelial cells, which can differentiate into invasive extravillous trophoblast (EVT) and multinucleated syncytiotrophoblast (STB). Trophoblast stem cells (TSC), derived from early first trimester placentas, have also been shown to be bipotential; however, their cell-of-origin has not been identified. In this study, we set out to probe the transcriptional diversity of early and late first trimester villous CTB (vCTB) and compare these to TSC. To this end, we performed single-cell RNA sequencing (scRNA-seq) on placental villous tissue from early (6-8 weeks) and late (12-14 weeks) first trimester placentas; we also evaluated CTB within basal (maternal) and chorionic (fetal) regions of early first trimester placenta, both by scRNA-seq and GeoMx-based spatial transcriptomics. Finally, we performed scRNA-seq on three TSC lines, derived from 6-8 week gestation placentas, as well as on early first trimester CTB at several timepoints during TSC derivation. We observed differences within CTB clusters associated with gestational age, further influenced by location near the basal or chorionic plates. We identified trophoblast states representing "initial state" vCTB (in vivo CTB progenitors), as well as additional CTB subtypes, precursor STB, and precursor and mature EVT. CTB progenitors appeared enriched in early first trimester placentas at the basal plate; additionally, basal plate CTB showed transcriptional features consistent with EVT bias, whereas chorionic plate CTB showed features associated with STB precursors. Clustering and trajectory inference analysis indicated that TSC were most like EVT precursor cells. In fact, vCTB lost their in vivo "initial state" markers, including PAGE4, as they transitioned to TSC during in vitro culture. This was confirmed by flow cytometric analysis of 6 different TSC lines, which showed uniform expression of proximal column markers ITGA2 and ITGA5. Additionally, we found that ITGA5+ CTB could be plated in 2D, readily differentiating only into EVT in the absence of lineage-directed cues, but failed to form self-renewing organoids; conversely, ITGA5- CTB could not be plated in 2D, but readily formed organoids. Our findings suggest that distinct CTB states exist in different regions of the placenta as early as six weeks gestation and that current TSC lines most closely resemble ITGA5+ proximal column trophoblast, biased toward the EVT lineage.