Crl 4003

The immortalized human endometrial stromal cell line (HESCs) was a gift from Professor Wang Haibin (ATCC® CRL-4003™) (Jiang et al. 2020 (link), Wei et al. 2022 (link)). The cells were grown in phenol red-free Dulbecco’s modified Eagle’s medium: nutrient mixture F-12 (DMEM/F12, Gibco) with 10% charcoal-stripped fetal bovine serum (CS-FBS, Biological Industries, Beit Haemek, Israel), 100 μg/mL streptomycin, 100 IU/mL penicillin, 1% insulin–transferrin–selenium solution (ThermoFisher Scientific), and 500 ng/mL puromycin (Sigma-Aldrich) at 37°C with 5% CO₂. The cell culture medium was exchanged every 48 h. For passaging, the cells were detached using trypsin with EDTA (Sigma) at 37°C for 3 min.

Endometrial biopsies were obtained from 7 pre-menopausal women with regular menstrual cycles (5 in proliferative phase and 2 in secretory phase) and from 1 peri-menopause woman, all undergoing histeroscopy for diagnostic purposes. The selected women did not show any evident endometrial pathology or suffer from any endocrine disorder or systemic disease and have not received any steroid treatment for at least 3 months prior to tissue collection. Uterine samples were obtained by Vacuum Aspiration Biopsy Random Assay (Vabra) (see Supplementary Information for the details of tissue culture preparation).
The immortalized human endometrial stromal cell line T-HESC, originated from normal human endometrial stromal cell immortalized with hTERT27 (link), was obtained from ATCC (CRL-4003^™). Cells were maintained in complete growth medium according to the manufacturer’s instructions. To induce T-HESC cell decidualization, stromal cells were stimulated with cAMP (0.5 mM; Sigma) and medroxyprogesterone acetate (MPA) (1 μM; Sigma) for 7 days.

Considering that both BMP2 and ICAM1 are expressed in human endometrial stromal cells throughout the menstrual cycle, and endometrial decidualization is a critical physiological event in the female menstrual cycle, thus immortalized human endometrial stromal cells (HESCs; ATCC^® CRL-4003) and primary HDSCs were used as study cell models, representing non-decidual and decidual stromal cells respectively, to systematically study the regulatory effect of BMP2 on ICAM1 in human endometrium at different stages of the menstrual cycle.

Primary endometrial stromal cells (ESCs) were isolated from the samples collected from the proliferative stage of the menstrual cycle by Pipelle biopsy from fertile, regularly cycling women and endometriosis-associated infertile women under anesthesia as described previously [22 (link)]. The cells were routinely incubated in Dulbecco’s Modified Eagle’s Medium (DMEM/F12) without phenol red, containing 10% activated carbon-adsorbed serum (BasalMedia, Shanghai, China). When performing in vitro decidualization, cells were incubated in DMEM/F12 without phenol red containing 2% carbon-adsorbed serum with medroxyprogesterone acetate (MPA, 100 nM, MCE, HY-B0469S) and 8-Bromo-cyclic adenosine monophosphate (8-Br-cAMP, 0.5 mM, Selleck, S7857) added to the cellular supernatant. After 2–6 d, cellular decidualization was assessed by evaluating the expression of decidualization marker genes and cell morphology.
Human endometrial stromal cells (ThESCs) were purchased from the American Type Culture Collection (CRL-4003; ATCC) and cultured in the same medium and environment as ESCs. For METTL3 intervention assays, ThESCs were pretreated with a METTL3-overexpressing vector (Dianjun, Shanghai, China), siRNAs of METTL3 (Dianjun, Shanghai, China), or their own negative control group for 24 h and then treated with MPA and 8-Br-cAMP for 4 d. The siRNA sequences are listed in Table S2.

Human primary ESCs were isolated from the normal endometrium of patients with tubal infertility without EMs. The tissues were minced with scissors and digested enzymatically with collagenase type II (0.1%; Sigma-Aldrich) for 40 min at 37°C with constant agitation. Then, the tissue pieces were sequentially filtered through sterile 400-µm and 100-µm sieve wires to remove undigested tissue and epithelial cells, and the filtrate was centrifuged at 129 g for 5 min to separate the ESCs from the collagenase. The ESC pellets were suspended in Red Blood Cell Lysis Buffer (C3702, Beyotime, Jiangsu, China) for 5 min to remove erythrocytes. The cell suspensions were centrifuged at 129 g for 5 min, and the pelleted ESCs were resuspended in Dulbecco’s Modified Eagle’s Medium (DMEM)/F-12 medium containing 10% fetal bovine serum (FBS). The ESCs were placed in a culture flask and incubated in 5% CO₂ at 37°C.
A human endometrial stromal cell line (ThESCs) was purchased from American Type Culture Collection (CRL-4003; ATCC) and cultured in the same medium and environment as the ESCs.

A human endometrial stromal cell line (hESC, CRL-4003) was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were seeded into 6-well plates at 2 × 10⁵ cells/well and cultured in Dulbecco’s modified Eagle’s medium: F12 without phenol red, supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic solution. Monolayers of confluent cells were scratched using a 100 μL pipette tip along a ruler without damaging the plastic and then washed twice with PBS to remove non-adherent cells. The medium was replaced with serum-free medium and the hESCs were cultured in six-well plates with inserts containing 2% human PRP or 2% serum [23 (link)]. We used an inverted microscope to monitor wound closure at 0, 6, 12, 18, and 24 h after the scratch and cell images were obtained at the same position. The wound areas in the cell migration assay were calculated using ImageJ (http://imageJ.nih.gov/ij/ accessed on 29 June 2020) [24 (link)].