Manifold roles of CCR7 chemokine GPCR in human trophoblast differentiation

CCR7 chemokine G protein-coupled receptor is expressed in extraembryonic tissues of the early human embryo, including trophectoderm and its derivatives cytotrophoblast (CTB), extravillous trophoblast (EVT), and syncytiotrophoblast (STB). However, its function in placental development remains poorly explored. Here, we generated human embryonic stem cells harboring CCR7 deletions and differentiated them into human trophoblast stem cell (hTSC) and hTSC-derived trophoblast organoids. We found that CCR7 mutant EVTs retained hTSC-like characteristics, exhibited decreased epithelial-to-mesenchymal transition, and reduced cell motility. Additionally, CCL21-CCR7 induced EVT terminal differentiation into endovascular EVT-like cells. Transcriptional profiling in CCR7 KO STBs identified reduced viral defense gene expression related to the protection against maternal-fetal transmission. Investigation of trophoblast organoids using single cell transcriptome profiling showed that CCR7 mutant trophoblast organoids comprised a smaller EVT population, but a larger STB population, compared to wild type organoids, while CellChat analysis indicated altered cell-cell communication including WNT, ACTIVIN, and IFN-I signaling pathways. Mechanistically, we found that CCR7 limited the cell-cell fusion of early STB differentiation by reducing cAMP levels. Together, our studies demonstrate that CCR7 plays multiple roles in cellular decision-making during trophoblast differentiation, promoting EVT differentiation, and limiting cell-cell fusion during early STB differentiation. Overall design: To understand the CCR7 function in trophoblast differentiation and maturation from naïve to hTSC as well as terminal differentiation of hTSC to EVT and STB, we generated CCR7 KO allele (A10) in the backbone of H9 hESCs and compared the RNA transcriptome of WT vs mutant in each cell types. Naïve H9 vs CCR7 KO (n=2), hTSC H9 vs CCR7 KO (n=4), EVT H9 vs CCR7 KO (n=4), and 3D STB H9 vs CCR7 KO (n=4)