Isolation and Culture of Human Decidual Stromal Cells

Primary HDSCs were separated from decidual tissues by means of enzymatic dispersion and mechanical dissociation, as mentioned before (Zhu et al., 2007 (link)). Generally, the samples were washed in cold phosphate-buffered saline (Gibco, Life Technologies, Inc., Carlsbad, CA, United States of America) three times, and then minced and treated with 0.1% collagenase (type IV; Sigma‒Aldrich), 0.1% hyaluronidase (type I-S; Sigma‒Aldrich) and 0.5 mg/ml DNase I (Sigma‒Aldrich) and subsequently digested in a shaking water bath for 60 min at 37°C. The supernatant was neutralized by the addition of phenol red-free DMEM/F12 medium supplemented with 10% FBS before the cells were passed through a 40 m nylon filter (BD Biosciences, Bedford, UK). The undigested tissue fragments were left on the filter, and the stromal cell-containing eluate was transferred into a 50 ml tube. The cells were then pelleted by centrifuging them at 1200 g for 3 min at room temperature. Following that, the cell pellets were washed, resuspended, and seeded in phenol red-free DMEM/F12 media with antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin, Life Technologies, Inc.), 10% FBS, 30 nM 17β estradiol (E2; Sigma Aldrich), and 1 μM progesterone (P4; Sigma Aldrich). All decidual stromal cell cultures were afterwards maintained at 37°C in a humid incubator with 5% CO2, unless otherwise stated, in this culture medium. The purity of the HDSCs was determined by immunofluorescent staining for vimentin and cytokeratin-7 as described previously (Zhu et al., 2007 (link)). HDSCs were cultivated at a density of 5 × 10⁵ cells per plate in 60-mm tissue culture dishes for the time- and concentration-dependent studies, and BMP2 was added in the same manner as HESCs.