Isolation and Decidualization of Human Endometrial Stromal Cells

Human endometrial stromal cells (HESCs) were isolated from endometrial tissues as described previously [76 (link)]. Purified HESCs were expanded in maintenance medium of DMEM/F-12 (Invitrogen, Schwerte, Germany) containing 10% dextran-coated charcoal-treated fetal bovine serum (DCC-FBS; Invitrogen, UK) and 1% antibiotic-antimycotic solution (Invitrogen). Confluent monolayers were decidualized in DMEM/F-12 containing 2% DCC-FBS with 0.5 mM 8-bromo-cAMP (8-Br-cAMP; Sigma, Munich, Germany) with or without 10⁻⁶ M medroxyprogesterone acetate (MPA; Sigma) to induce a differentiated phenotype. Where indicated, the cells were treated with recombinant LEFTY2 (25 ng/ml; R&D Systems, Germany) as described previously [77 (link)]. Ionomycin was used at 1 μM (Sigma) and the Orai inhibitors: 2-APB, YM-58483, and MRS-1845(TOCRIS, Germany). Ishikawa cells, an endometrial epithelial-like cell line (ECACC 99040201) [28 (link), 29 (link)], were maintained in DMEM/F12 (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 2 mM L-glutamine, and 100 U/ml penicillin/streptomycin (Invitrogen). All cells were incubated at 37 °C in a humid atmosphere maintained at 5% (vol/vol) CO₂, and routinely tested for mycoplasma infection.