Single-cell transcriptomics reveal differences between chorionic and basal plate cytotrophoblasts and 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 placentae, have also been shown to be bipotential. In this study, we set out to probe the transcriptional diversity of first trimester CTB and compare TSC to various subgroups of CTB. We performed single-cell RNA sequencing on six normal placentae, four from early (6-8 weeks) and two from late (12-14 weeks) first trimester, of which two of the early first trimester cases were separated into basal (maternal) and chorionic (fetal) fractions prior to sequencing. We also sequenced three TSC lines, derived from 6-8 week placentae, to evaluate similarities and differences between primary CTB and TSC. CTB clusters displayed notable distinctions based on gestational age, with early first trimester placentae showing enrichment for specific CTB subtypes, further influenced by origin from the basal or chorionic plate. Differential expression analysis of CTB from basal versus chorionic plate highlighted pathways associated with proliferation, unfolded protein response, and oxidative phosphorylation. We identified trophoblast states representing initial progenitor CTB, precursor STB, precursor and mature EVT, and multiple CTB subtypes. CTB progenitors were enriched in early first trimester placentae, with basal plate cells biased toward EVT, and chorionic plate cells toward STB, precursors. Clustering and trajectory inference analysis indicated that TSC were most like EVT precursor cells, with only a small percentage of TSC on the pre-STB differentiation trajectory. 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, forming only EVT upon spontaneous differentiation, 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+ CTB, biased toward the EVT lineage.