Small extracellular vesicles (sEVs) are important mediators of cell-to-cell communication involved in the successful establishment of a pregnancy. Human decidual stromal cells play a key role in regulating trophoblast invasion. Nevertheless, the regulatory functions of decidual stromal cells-derived sEVs in human trophoblast cells are still unclear. In this study, primary human decidual stromal cells were isolated, and immortalized human endometrial stromal cell line (HESCs) were decidualized into human decidual stromal cells (HDSCs) using hormonal cocktail containing medroxy progesterone 17-acetate (MPA), estrogen and cAMP analog. HDSC-sEVs were isolated from both primary human decidual stromal cells and immortal HDSCs, respectively, and identified by transmission electron microscopy and western blotting. EV uptake assay indicated that HDSC-sEVs could be uptaken by trophoblast cells. HDSC-sEVs could increase the invasiveness and the expression level of N-cadherin of trophoblast cells with elevated phosphorylation of SMAD2 and SMAD3 in the cells. Silencing of N-cadherin could block cell invasion induced by HDSC-sEVs, while knockdown of SMAD2 and SMAD3 could inhibit the upregulation of N-cadherin in trophoblast cells. Taken together, our results suggested a regulatory effect of HDSC-sEVs in the invasion of trophoblast cells, and HDSC-sEVs may be important mediators of trophoblasts during embryo implantation and placentation.
Supplemental FIGURE 1 Primary human EVTs uptake primary HDSC-sEVs (A) The purified DSCs were determined by positive staining of Vimentin and negative staining of CK-7. The purity of DSC cells was ∼95%. Scale bar: 50μm. (B)Primary human extravillous trophoblast cells (EVTs) were identified by immunofluorescence staining of CK7. Scale bar: 50 μm. (C) The morphology of primary HDSC-sEVs from primary decidual stromal cells. Scale bar: 200nm. (D) The expression of positive markers (Alix, CD63 and CD81) of primary HDSC-sEVs were detected by Western blotting. (E) Primary EVTs were pre-treated with PKH67-labeled primary HDSC-sEVs for 4 hours following by DAPI (blue) staining for confocal microscopy analysis. Scale bar, 10μm.