Foetal bovine serum (fbs)

Endometrial stromal cells were isolated from ectopic, eutopic, and normal control endometrial tissues and were named ESCs, EuSCs, and NESCs, respectively, as described in a previous study [21 (link)]. Samples were washed twice with phosphate-buffered saline (PBS), minced, and coated with 1 mg/ml collagenase type IV (BS165, Biosharp, The Netherlands). Samples were incubated at 37 °C with mild shaking for two hours in a culture flask. Next, a 40-mm sieve was used to remove debris and other cells, such as endometrial epithelial cells, and the solution that passed through was centrifuged for 10 min at 1000 r/min. Cell precipitates were collected by removing the supernatant. The cells were grown in a 5% CO₂ humidified atmosphere at 37 °C in DMEM/F12 (MA0214, Meilunbio, China) supplemented with 10% foetal bovine serum (BI, Israel).

Human trophoblast cell lines (Jeg3, HTR8) and human decidual stromal cell line (hESC) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Jeg3 and HTR8 cells were cultured in a 5% humidified carbon dioxide atmosphere at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 (Gibco, Life Technologies, Grand Island, NY, USA) with 10% foetal bovine serum (Gibco, Life Technologies, Grand Island, NY, USA), 50 mg/mL streptomycin, and 50 U/mL penicillin. Jeg3 is the only trophoblast cell line that expresses human leukocyte antigen G (HLA-G), which is gradually expressed during development from CTBs to EVTs.
hESCs were cultured in a 5% humidified carbon dioxide atmosphere at 37 °C in phenol red-free Dulbecco’s modified Eagle’s medium (DMEM)/F-12 (Meilunbio, Dalian, China) with 10% foetal bovine serum, 50 mg/mL streptomycin, and 50 U/mL penicillin. One micromolar medroxyprogesterone-17-acetate (MPA) (HY-B0469, MedChemExpress, NJ, USA) and 0.5 mM N6,20-O-dibutyryladenosine cAMP sodium salt (db-cAMP) (HY-B0764, MedChemExpress, Monmouth Junction, NJ, USA) were added to the culture for 6 days to induce hESC decidualization in vitro [14 (link)].