Regionally distinct trophoblast regulate barrier function and invasion in the human placenta
- The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell, University of California, San Francisco, United States
- Department of Urology, University of California, San Francisco, United States
- Center for Reproductive Sciences, University of California, San Francisco, United States
- Developmental and Stem Cell Biology Graduate Program, University of California, San Francisco, United States
- Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco, United States
Figures
Figure 1 with 8 supplements
The transcriptional landscape of the villous (VC) and smooth chorion (SC) at mid-gestation.
(a) Left: Schematic of the placenta at mid-gestation, highlighting the regions sampled, together with the methods used for cell isolation and characterization. Right: Schematic of the cell types and …
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Figure 1—source data 1
Marker genes for the clusters of the Immune subset.
- https://cdn.elifesciences.org/articles/78829/elife-78829-fig1-data1-v2.csv
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Figure 1—source data 2
Marker genes for the clusters of the Stroma subset.
- https://cdn.elifesciences.org/articles/78829/elife-78829-fig1-data2-v2.csv
Figure 1—figure supplement 1
Metrics of the integrated dataset.
(a) Violin plots of the number of unique genes (left), number of UMI (middle), and the percent of mitochondrial reads (right) per cell for each broad cell type cluster. (b) The total number of cells …
Figure 1—figure supplement 2
XIST expression by sample.
(a) Violin plot of XIST transcript expression in each sample for each trophoblast cluster. Since all trophoblast are of fetal origin, samples expressing XIST are XX and samples with no XIST …
Figure 1—figure supplement 3
Comparison of stroma and immune clusters to Vento-Tormo et al., 2018.
(a) Spearman correlations between the average expression within each stromal cell cluster identified in this study (y-axis) and in Vento-Tormo et al., 2018 (x-axis). Black dots denote p-value ≤ 0.05 …
Figure 1—figure supplement 4
Comparison of stroma and immune clusters to Pique-Regi et al., 2019.
(a) Spearman correlations between the average expression within each stromal cell cluster identified in this study (y-axis) and in Pique-Regi et al., 2019 (x-axis). Black dots denote p-value ≤ 0.05 …
Figure 1—figure supplement 5
Metrics and markers of the immune cell subset.
(a) UMAP of all subclustered immune cells (n=14,805). Colors correspond to the clusters in the legend at the right. (b) Heatmap of selected marker genes of each immune cell cluster. Expression was …
Figure 1—figure supplement 6
Independent analysis of villous chorion (VC) and smooth chorion (SC) immune cells.
(a) UMAP of subclustered stromal cells isolated from the VC. Colors correspond to the clusters at the right. (b) UMAP of subclustered stromal cells isolated from the SC. Colors correspond to the …
Figure 1—figure supplement 7
Metrics and markers of the stromal cell subset.
(a) UMAP of subclustered stromal cells (n=3883). Colors correspond to the clusters in the legend at the right. (b) Heatmap of selected marker genes for each stromal cell cluster. Expression was …
Figure 1—figure supplement 8
Independent analysis of villous (VC) and smooth chorion (SC) stromal cells.
(a) UMAP of subclustered immune cells isolated from the VC. Colors correspond to the clusters at the right. (b) UMAP of subclustered immune cells isolated from the SC. Colors correspond to the …
Figure 2 with 4 supplements
Identification of a smooth chorion-specific cytotrophoblast.
(a) UMAP of subclustered trophoblasts (n=29,668). Colors correspond to the clusters at the right. (b) Dot plot showing average expression and percent of cells in each cluster as identified by the …
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Figure 2—source data 1
Marker genes for each cluster in the trophoblast subset.
- https://cdn.elifesciences.org/articles/78829/elife-78829-fig2-data1-v2.csv
Figure 2—figure supplement 1
Metrics of the trophoblast subset.
(a) Violin plots of the number of unique genes (left), number of UMI (middle), and the percent of mitochondrial reads (right) per cell for each trophoblast cluster. (b) UMAPs of the trophoblast …
Figure 2—figure supplement 2
Markers of the trophoblast subset.
(a) Heatmap of the expression of the top 20 marker genes for each trophoblast population. (b) Expression of phasic transcripts MKI67 (top), PCNA (middle), and TOP2A (bottom) were projected in UMAP …
Figure 2—figure supplement 3
Independent analysis of villous (VC) and smooth chorion (SC) trophoblast.
(a) UMAP of subclustered trophoblasts isolated from the VC. Colors correspond to the clusters at the right. (b) UMAP of subclustered trophoblasts isolated from the SC. Colors correspond to the …
Figure 2—figure supplement 4
KRT6 expression in the villous chorion (VC) region.
(a) Violin plot of CDH1 transcript expression across all trophoblast clusters. (b) Immunofluorescence co-localization of CDH1 and PAGE4 in the VC (left) and SC (right). (c) Immunofluorescence …
Figure 3 with 2 supplements
A common cytotrophoblast (CTB) progenitor gives rise to syncytiotrophoblasts (STBs) in the villous chorion (VC) and smooth chorion (SC)-CTBs in the SC.
RNA velocity vector projections overlaid on to UMAPs for trophoblast cells isolated from the (a) VC and (b) SC. Arrows denote direction and magnitude is represented by line thickness. (c) Pseudotime …
Figure 3—figure supplement 1
Smooth chorion (SC) trophoblast display reduced expression of syncytiotrophoblast (STB), increased expression of epithelial TFs, and proliferate.
(a) Violin plots of expression of markers of STB differentiation in each trophoblast cluster shown by region, demonstrating reduced expression of all markers in STB Precursor and STB clusters. (b) …
Figure 3—figure supplement 2
Predicted interactions between cytotrophoblasts (CTBs).
(a) Predicted receptor-ligand interactions from CellPhoneDB between CTB clusters of the villous chorion (VC) for interactions which are unique to the VC. (b) Predicted receptor-ligand interactions …
Figure 4 with 4 supplements
Smooth chorion-specific cytotrophoblasts (SC-CTBs) express a distinct epidermal transcriptional program.
(a) Heatmap of gene ontology analysis adjusted p-values. Dark red corresponds to the lowest p-values and white represents p-values greater than 0.0005. Ontology categories are organized by …
Figure 4—figure supplement 1
Complete cytotrophoblast (CTB) gene ontology analysis.
The unabridged gene ontology results from each CTB cluster, displayed as adjusted p-values. Dark red corresponds to the lowest p-values and white represents p-values greater than 0.0005. Ontology …
Figure 4—figure supplement 2
Similarities and differences between cytotrophoblast (CTB) 1 in villous chorion (VC) and SC.
(a) Immunofluorescence co-localization of CDH1 and non-phosphorylated CTNNB1 in the VC (left) and SC (right), showing identical domains of expression in both regions. For all images, nuclei were …
Figure 4—figure supplement 3
Cytokeratin expression in villous (VC) and smooth chorion (SC) trophoblast.
(a) Violin plots of cytokeratins expressed in trophoblast in both VC and SC regions by cluster and region of origin. (b) Violin plots of cytokeratins expressed in SC trophoblast by cluster and …
Figure 4—figure supplement 4
IFITM3 expression in cytotrophoblast (CTB) populations.
Violin plot of IFITM3 expression in trophoblast clusters by region, showing increased expression in smooth chorion (SC) compared to villous chorion (VC).
Figure 5 with 3 supplements
Extravillous trophoblasts (EVTs) of the villous chorion (VC) and SC regions display distinct invasive activity but are transcriptionally similar.
(a) Expression of HLA-G transcript per cell projected in UMAP space for the VC (left) and SC (right). Expression ranged from low in light gray to high in dark red. (b) Immunofluorescence …
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Figure 5—source data 1
EVT 1 cluster differentially expressed genes between regions.
- https://cdn.elifesciences.org/articles/78829/elife-78829-fig5-data1-v2.csv
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Figure 5—source data 2
EVT 2 cluster differentially expressed genes between regions.
- https://cdn.elifesciences.org/articles/78829/elife-78829-fig5-data2-v2.csv
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Figure 5—source data 3
EVT 3 cluster differentially expressed genes between regions.
- https://cdn.elifesciences.org/articles/78829/elife-78829-fig5-data3-v2.csv
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Figure 5—source data 4
EVT 4 cluster differentially expressed genes between regions.
- https://cdn.elifesciences.org/articles/78829/elife-78829-fig5-data4-v2.csv
Figure 5—figure supplement 1
Functional annotation of extravillous trophoblast (EVT) clusters.
(a) Gene ontology results from each EVT cluster displayed as adjusted p-values. Dark red corresponds to the lowest p-values and white represents p-values greater than 0.0005. Ontology categories are …
Figure 5—figure supplement 2
Differential expression between extravillous trophoblasts (EVT) from villous (VC) and smooth chorion (SC).
(a) Volcano plots showing the differentially expressed genes the VC or SC for each EVT cluster. Only significantly differentially expressed genes are plotted (p-value < 0.05). Genes with higher …
Figure 5—figure supplement 3
Predicted interactions between extravillous trophoblast (EVT) and stromal cells.
(a) Predicted receptor-ligand interactions from CellPhoneDB between EVT-Stromal cells of the villous chorion (VC) for interactions which are unique to the VC. (b) Predicted receptor-ligand …
Figure 6 with 4 supplements
Cytotrophoblasts (CTBs) of the SC inhibit extravillous trophoblast (EVT) invasion.
(a) Immunofluorescence co-localization of np-CTNNB1 and HLA-G in the VC. (b) Immunofluorescence co-localization of KRT6 and HLA-G in the SC. Arrowheads denote CTB and EVT interactions. (c) Schematic …
Figure 6—figure supplement 1
Cytotrophoblast (CTB)-extravillous trophoblasts (EVT) interactions in the villous chorion (VC) or SC region.
Co-localization of KRT6 (SC-CTB marker) and HLA-G (EVT marker) showing rare KRT6+ cells in the VC (top). These cells are few and do not interact with EVT in the same manner as was observed in the …
Figure 6—figure supplement 2
Representative images of the transwell invasion assay.
Representative images of trophoblast projections through the transwell filter are seen as white dots. For quantification, the number of projections (white bright dots) was counted. For each sample, …
Figure 6—figure supplement 3
Cell density is not correlated with culture in conditioned media.
The DAPI+ area in each field of view was quantified and plotted as a measure of cell density. Each dot represents the percent area of the field of view which stained with DAPI. Measurements from …
Figure 6—figure supplement 4
Complete predicted interactions between cytotrophoblast (CTB)-extravillous trophoblast (EVT) in the smooth chorion (SC).
(a) Predicted receptor-ligand interactions from CellPhoneDB between CTB-EVT and the SC. The strength of interaction is estimated by mean expression and is plotted in the heatmaps. Receptor-ligand …
Tables
Key resources table
| Reagent type(species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Rat anti E-cadherin Monoclonal Antibody (ECCD-2) | Thermofisher Scientific | 13-1900 | IF (1:250) |
| Antibody | Rabbit Anti-PAGE4 antibody (Polyclonal) | Sigma-Aldrich | HPA023880 | IF (1:100) |
| Antibody | Rabbit Recombinant Anti-Cytokeratin 6 Monoclonal antibody [EPR1603Y] | Abcam | ab52620 | IF (1:100) |
| Antibody | Rabbit anti Non-phospho (Active) β-Catenin (Ser33/37/Thr41) Monoclonal Antibody | Cell Signaling | 8814 | IF (1:100) |
| Antibody | Mouse anti Cytokeratin 14 Monoclonal Antibody (LL002) | Invitrogen | MA5-11599 | IF (1:100) with Antigen Retrieval |
| Antibody | Rabbit Anti-KLF4 antibody (polyclonal) | Sigma-Aldrich | HPA00292 | IF (1:100) |
| Antibody | Rat anti Cytokeratin (7D3) Monoclonal Antibody | Susan Fisher/University of California, San Francisco Cat# Fisher_001-clone7D3,RRID:AB_2631235 | AB_2631235 | IF (1:100) |
| Antibody | Mouse anti HLA-G (4H84) Monoclonal Antibody | Susan Fisher/University of California, San Francisco Cat# Fisher_002-clone4H84, RRID:AB_2631236 | AB_2631236 | IF (1:20) with Antigen Retrieval |
| Software, algorithm | R | https://www.r-project.org/ | ||
| Software, algorithm | ImageJ | ImageJ (http://imagej.nih.gov/ij/) | ||
| Software, algorithm | Seurat (3.1.3) | https://satijalab.org/seurat/ | ||
| Software, algorithm | cellranger (3.0.2) | https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/feature-bc | ||
| Software, algorithm | ClusterProfiler | https://guangchuangyu.github.io/software/clusterProfiler/ | ||
| Software, algorithm | scVelo | https://github.com/theislab/scvelo (Marsh, 2022b) copy archived at swh:1:rev:1805ab4a72d3f34496f0ef246500a159f619d3a2 | ||
| Software, algorithm | Prism 6.0 | https://www.graphpad.com/scientific-software/prism/ | ||
| Software, algorithm | DoubletFinder | https://github.com/chris-mcginnis-ucsf/DoubletFinder (Marsh, 2022c) copy archived at swh:1:rev:67fb8b5808eb16167ead5f9b439677cc24837554 |
Additional files
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Supplementary file 1
Number of cells captured from each region per cluster for the trophoblast dataset.
- https://cdn.elifesciences.org/articles/78829/elife-78829-supp1-v2.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/78829/elife-78829-mdarchecklist1-v2.docx
