Immortalized Human Endometrial Stromal Cell Decidualization

The human ESC-derived telomerase-immortalised cell line, St-T1b (Samalecos et al., 2009 (link)), kindly provided by Professor Jan Brosens (University of Warwick, UK), was maintained in phenol red-free Dulbecco's modified Eagle medium DMEM/Ham's F12 (DMEM/F12; Invitrogen, Renfrew, UK) with 10% steroid-depleted foetal calf serum (FCS) supplemented with 2 mM L-glutamine, 1 μg/ml insulin, 0.3 ng/ml 17β-oestradiol (E2), 50 μg/ml penicillin, 50 μg/ml streptomycin, and 0.2% Primocin (Invivogen, Toulouse, France) (ESC medium) at 37°C in an atmosphere of 5% CO₂. Phenol red-free medium was used in all experiments, due to phenol red’s known oestrogenic activity (Berthois et al., 1986 (link)). To induce decidualisation, cells were treated with minimal medium 1 (MM1; ESC medium without insulin and E2) containing increasing concentrations of the progestin, medroxyprogesterone acetate (MPA), 8-bromoadenosine 3',5'-cyclic adenosine monophosphate (8-Br-cAMP; Cambridge Bioscience, Cambridge, UK) and E2, or MM1 with 0.001% ethanol (Table I), every 48 h, and cultured over 8 days at 37°C in an atmosphere of 5% CO₂. All reagents for St-T1b cell culture were purchased from Sigma-Aldrich (Sigma-Aldrich, Dorset, UK) unless stated otherwise. Cultured cells were fixed in 4% paraformaldehyde (PFA) on days 4, 6, and 8 for immunocytochemical analysis.