Isolation and culture of EnSCs

EnSC isolation was performed as described previously [19]. In brief, the fresh endometrial tissue samples were rinsed with PBS and minced into small pieces (1 mm³) with sterile scissors. The tissue fragments were digested with type I collagenase [1 mg/mL (Sigma, USA), diluted in DMEM/F12 medium (HyClone, USA)] for 60 min at 37 °C with shaking every 10 min, and the tissue digests were successively filtered through 100 μm and 40 μm cell strainers (Corning, New York, USA). Then, the cell suspension was washed twice with PBS and gently resuspended in growth medium [DMEM/F12 supplemented with 10% FBS (Gibco, USA) and 1% penicillin/streptomycin (P/S, Life Technologies, USA)], seeded into 25 cm² cell culture flasks and incubated at 37 °C in a humidified atmosphere containing 5% CO₂. After 2 days of incubation, the non-adherent cells were washed away leaving behind the adherent cells that were growing as fibroblastic cells in clusters. The medium was replaced every 3 days. At 80–90% confluence (P0), the cells were detached by treatment with 0.25% trypsin/1 mM EDTA and subcultured into new flasks at a ratio of 1:2. Finally, all EnSC samples were cryopreserved using standard methods in complete growth medium containing 10% DMSO for the subsequent experiments.