Figure 6. 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 of the transwell invasion assay (top). Cells from either chorionic region were plated in the upper chamber of the transwell. After 39 hr of culture, the transwell membrane was fixed and stained with a Pan-cytokeratin antibody. The projections through the membrane are denoted by black arrowheads and quantified below. Results from the VC derived cells are shown in black, and the results from the SC derived cells are shown in blue. (d) Results from three biological replicates from each chorionic region cultured with conditioned medium from either VC or SC cells. The gestational ages of the plated cells are shown adjacent to the y-axis. The gestational ages of the cells from which conditioned medium was collected are noted in the legends at the right. The results for cells cultured with VC cell conditioned medium are denoted by black dots, and the results for those cultured in SC cell conditioned medium are denoted by open squares. p-Values were determined by t-test and are listed above each comparison. (e) Predicted receptor-ligand interactions from CellPhoneDB. The strength of interaction is estimated by mean expression and is plotted in the heatmap. Receptor-ligand interactions and cell pairs are listed such that Molecule 1 is expressed by Cell 1 and Molecule 2 is expressed by Cell 2. For all images, nuclei were visualized by DAPI staining; scale bar = 100 μm. Abbreviations: AV = anchoring villi; FV = floating villi; SC = smooth chorion epithelium; Dec. = decidua.

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 SC. Co-localization of non-phosphorylated CTNNB1 (CTB 1 marker) and HLA-G (EVT marker) in the SC (bottom). Limited interactions between these populations was observed. The basal lamina separating the fetal stroma from the SC epithelium is marked by the white dashed line. For all images, nuclei were visualized by DAPI stain; scale bar = 100 μm. Abbreviations: AV = anchoring villi; FV = floating villi; SC = smooth chorion epithelium; Dec. = decidua.

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, the median DAPI area was quantified across the transwell membrane, and then the projections multiplied by a factor to normalize for cell density. Scale bar = 100 μm.

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 each experiment are shown and the gestational ages of the cultured cells is on the y-axis at the left. Measurements of cells treated with control media (Serum Free Media) are shown in red, VC cell conditioned media are shown in black, and smooth chorion (SC) cell conditioned media are shown in open squares.

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 interactions and cell pairs are listed such that Molecule 1 is expressed by Cell 1 and Molecule 2 is expressed by Cell 2.