Single-cell RNA-seq reveals the diversity of trophoblast subtypes and patterns of differentiation in the human placenta

Abstract

The placenta is crucial for a successful pregnancy and the health of both the fetus and the pregnant woman. However, how the human trophoblast lineage is regulated, including the categorization of the placental cell subtypes is poorly understood. Here we performed single-cell RNA sequencing (RNA-seq) on sorted placental cells from first- and second-trimester human placentas. New subtypes of cells of the known cytotrophoblast cells (CTBs), extravillous trophoblast cells (EVTs), Hofbauer cells, and mesenchymal stromal cells were identified and cell-type-specific gene signatures were defined. Functionally, this study revealed many previously unknown functions of the human placenta. Notably, 102 polypeptide hormone genes were found to be expressed by various subtypes of placental cells, which suggests a complex and significant role of these hormones in regulating fetal growth and adaptations of maternal physiology to pregnancy. These results document human placental trophoblast differentiation at single-cell resolution and thus advance our understanding of human placentation during the early stage of pregnancy.

Fig. 1

Isolation and validation of placental cells from 8 W and 24 W human placentas for single-cell RNA-seq. a Diagram of the human placental villi showing the localization of each cell type involved in this study and the markers used to sort these cells. CTB_8W, STR_8W, STB_8W, and EVT_8W: cytotrophoblasts, stromal cells, syncytiotrophoblast and extravillous trophoblasts sorted from the 8 week placental villi, respectively; EVT_24W, extravillous trophoblasts sorted from 24 week decidua. b Representative images showing bright field (upper panel) and immunofluorescence staining of STBs (bottom panel; red arrowhead). The beta subunit of chorionic gonadotropin (CGB) is a canonical marker for the STB. DAPI staining shows the nuclei (here and after). c Dot plot showing the expression of canonical marker genes for the defined cell types. d Box plot of the gene numbers detected in the defined cell types. e Box plot showing the distribution of the ratio of reads mapping to mitochondria in each cell type. f T-SNE distributions of single cells from the 7 defined cell types sorted by MACS from the 8 W and 24 W placentas

Fig. 2

Three CTB subtypes were identified in the first trimester human placenta. a Heatmap showing the expression of cell cycle-related genes in the 3 different CTB subtypes. Cells expressing over 100 cell cycle-related genes were defined as cycling cells. b Violin plot displaying the cell cycle-related marker genes for CTB_8W cells. c Immunohistochemistry staining of human first trimester placental villi using the indicated antibodies. The arrows indicate RRM2-positive CTB cells, and the arrowheads indicate RRM2-negative CTB cells. d GO analysis of the DEGs for the three different subtypes of CTB_8W cells. e Dot plot showing the expression of the indicated genes in the indicated subtypes of cells. f Localization of Syncytin-2 in human placental villi from 8 weeks of pregnancy by immunofluorescence staining. The white arrowheads indicate CTB_8W_1 cells. DAPI staining shows the nuclei. STB, syncytiotrophoblast layer (the area between white and yellow dotted lines); CTB, cytotrophoblast cells. g Representative still images corresponding to Supplementary information, movie S1, time-lapse imaging of the fusion process of 293 T cells mediated by Syncytin-2. The arrowheads indicate two single cells (transfected with Syncytin-2, together with either EGFP or mCherry) about to undergo fusion. The arrow indicates the fusion site. The circled dashed lines indicate the fused cells

Fig. 3

EVT subtypes and differentiation of CTBs into EVTs in the first and second trimesters. a Dot plot showing candidate marker genes for the indicated cell subtypes. b Immunohistochemistry staining of HLA-G and TAC3 in the decidua of 24 W human placenta. Arrows, EVTs with high expression of TAC3; arrowheads, EVTs with low expression of TAC3. c GO analysis of two subtypes of EVT_24W cells. d Pseudotime analysis of the indicated cells. Component 2 displays the differentiation of the CTB and EVT cells over time. e, f Top 10 transcription factors regulating the differentiation from CTB_8W to EVT_8W, and from EVT_8W to EVT_24W cells. Blue and red, the repressed and activated targets for each TF, respectively

Fig. 4

Identification of new subtypes of macrophages and mesenchymal stromal cells from the first trimester villous stromal core. a GO analysis of two subtypes of mesenchymal stromal cells. b Line plot showing the candidate marker genes for Mes_1 and Mes_2 cells. c Immunofluorescence staining of the indicated molecules in the 8 week human placental villi. The white arrows and arrowheads indicate mesenchymal stromal cells with high or low expression of DLK1, respectively. d GO analysis of two subtypes of macrophages. e Line plot showing the candidate marker genes for Macro_1 and Macro_2. f Immunofluorescence staining of the indicated molecules in the 8 week human placental villi. The white arrow and arrowhead indicate macrophages with high and low expression of MRC1, respectively

Fig. 5

Expression of epigenetic modification-related genes and imprinted genes in the 14 cell subtypes. a Heatmap of the genes associated with DNA methylation and demethylation. b Immunofluorescence staining of the indicated molecules in the human placental villi at 8 weeks of pregnancy. The cells between the white and yellow dashed lines are CTBs. STR, stromal cells. c, d Heatmaps of the chromatin modification writers (c) and erasers (d) in each cell subtype

Fig. 6

Heatmap of polypeptide hormone genes expressed in the 14 cell subtypes

Fig. 7

Summary of newly found cell subtypes and their marker genes in the human placenta. Note: the known populations of cells, including endothelial cells and En-EVT, which were not included in this study, are in grey