Single-cell transcriptomics reveal differences between chorionic and basal plate cytotrophoblasts and trophoblast stem cells

Abstract

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.

Fig. 1.

Integration and annotation of early and late first trimester cells. (A) UMAP of all placental cells following quality control filtering, integration, and clustering. (B) Dot plot and hierarchical clustering of clusters using trophoblast and non-trophoblast specific gene expression. (C) Dot plot and hierarchical clustering of clusters using cell type specific gene expression markers. (D) UMAP of trophoblast cells only annotated based on cell type specific gene expression shown in part (C). (E) UMAPs depicting the cell density of the cells in early first trimester (left) and late first trimester (right) placentae.

Fig. 2.

Trajectory inference of first trimester trophoblast cells. (A) RNA velocity projected as streamlines on the integrated first trimester trophoblast UMAP. (B) Initial state as calculated by CellRank. (C) Expression of the initial state markers, HMMR, TROAP, and LY6E on the integrated first trimester trophoblast UMAP.

Fig. 3.

Basal and chorionic plate trajectory inference and initial state. (A) UMAP of annotated and integrated basal and chorionic plate cells from two early first trimester placentae (left). Basal and chorionic plate cells were sequenced from each placenta. When integrated and clustered, basal and chorionic plate cells segregated within each cluster. Location of basal (dark blue) and chorionic (light blue) cells are shown (right). (Bottom) Table shows the number of cells originating from either the basal or chorionic plate making up each cluster. (B) RNA velocity projected as streamlines on the integrated basal and chorionic plate UMAP (left). CellRank calculated initial state locations colored by CTB cluster color (right). (C) Expression of the initial state makers, HMMR, TROAP, and LY6E on the integrated basal and chorionic plate UMAP. (D) In-situ hybridization of the initial state markers, HMMR, TROAP, and LY6E in a 6-week placenta, showing expression in a subgroup of villous CTB (arrowheads) within the gestational sac (“sac”), as well as in chorionic villi (“v”) near the chorionic and basal plates, and within the proximal column trophoblast (circles). Scale bar=125 μm.

Fig. 4.

Annotation and trajectory inference of integrated TSC and first trimester trophoblast. (A) UMAP of integrated trophoblast and TSC annotated based on cell type specific gene expression as shown in Figure S6C. (B) Differentially expressed genes in TSC cluster compared to placental clusters in early first trimester placentae and TSC integrated UMAPs. (C) In-situ hybridization of PAGE4 in a 6-week placenta, showing uniform expression in villous CTB (arrowheads) within the gestational sac (“sac”), as well as in chorionic villi (“v”) near the chorionic and basal plates, but excluded from proximal column trophoblast (circles). Scale bar=125 μm. (D) RNA velocity projected as streamlines on the integrated early first trimester placentae and TSC integrated UMAP after the late first trimester placental cells were removed. (E) CellRank calculated initial state location in early placentae and TSC integrated UMAP. (F) Violin/box plots of the initial state genes HMMR and TROAP in each cluster of the integrated early placentae and TSC data.

Fig. 5.

TSC phenotype and differentiation potential compared to primary CTB. (A) Percentage of TSC (undifferentiated state) expressing various markers of CTB and EVT by flow cytometry. Data are reported as mean ± standard deviation of 6 distinct TSC lines. (B) Percentage of first trimester CTB expressing the indicated surface marker by flow cytometry, at isolation and after 4 days of in vitro culture. Data are reported as mean ± standard deviation (n=2). (C) Quantitative PCR of STB marker CGB, and EVT markers ASCL2 and ITGA1, in TSC spontaneously differentiated over four days in normoxia (N, 21% oxygen) or hypoxia (H, 2% oxygen). Data are presented as fold change over undifferentiated TSC, reported as mean ± standard deviation (n=2) with p-values calculated using one-way ANOVA. (D) Primary first trimester CTB MACS-sorted based on ITGA5 expression, allowed to spontaneously differentiate over 4 days in either normoxia (21% oxygen) or hypoxia (2% oxygen), then fixed and stained for EVT (HLA-G) and STB (SDC1, CYP19A1) markers, and DAPI. Only ITGA5⁺ cells are shown, as ITGA5⁻ cells did not adhere to any substrate in 2D. (E) Organoid (T-Org) formation using first trimester CTB sorted for ITGA5. Table shows number of successful attempts to derive and culture organoids beyond 5 passages, starting from first trimester CTB MACS-sorted based on ITGA5 expression. Brightfield images of representative T-Org, derived from ITGA5⁻ and ITGA5⁺ CTB.