The endometrial tissues were processed for stromal cell culture as described previously (Fernandez-Shaw et al., 1992 (link)). Briefly, the samples were suspended in PBS after washing twice in 0.9% normal saline. The tissues were then washed twice in DMEM/F12, chopped and digested with 0.2% collagenase type I (c0130; Sigma, St. Louis. MO, United States) for 50 min at 37°C and finally digested with 0.1% deoxyribonuclease (DN25; Sigma) for 20 min at 37°C. The suspension was filtered through a 40 μm griddle (CLS431750-50EA; Sigma, United States), which permits only stromal cells to pass. Primary ESCs were cultured in Phenol Red-free Dulbecco’s modified Eagle’s medium (DMEM)/F-12 (Gibco, Grand Island, NY, United States) plus 10% charcoal-stripped fetal bovine serum (Biological Industries, US origin) and 1% penicillin-streptomycin-neomycin antibiotic mixture (Thermo Fisher Scientific) at 37°C with a 5% CO2 atmosphere. Cell purity was tested routinely by immunofluorescence staining for cytokeratin and vimentin.
To induce decidualization in vitro, 3 × 105 ESCs were incubated with DMEM/F12 containing 2% charcoal-stripped FBS, 1 μmol/L medroxyprogesterone-17-acetate (MPA) (Sigma) and 0.5 mmol/L N6, 20-O-dibutyryladenosine cAMP sodium salt (db-cAMP) (Sigma) for 4 days, and the medium was changed every other day.
To induce decidualization in vitro, 3 × 105 ESCs were incubated with DMEM/F12 containing 2% charcoal-stripped FBS, 1 μmol/L medroxyprogesterone-17-acetate (MPA) (Sigma) and 0.5 mmol/L N6, 20-O-dibutyryladenosine cAMP sodium salt (db-cAMP) (Sigma) for 4 days, and the medium was changed every other day.