8 bromoadenosine 3 5 cyclic monophosphate

Induced pluripotent stem cells were seeded onto flasks coated with Matrigel at a density of 0.5–1 × 10⁴ cells per cm² in primed hiPS cell medium (KSR/FGF2). After 48 h, the medium was changed daily with RPMI-based medium with B27 supplement (Gibco, ThermoFisher Scientific), 100 ng ml⁻¹ activin A (Peprotech), 1 µM CHIR99021, 1% penicillin and streptomycin for 3 days. On days 4–8, the medium was changed daily with DMEM/F12-based medium with N2 (Gibco, ThermoFisher Scientific) and B27 supplements, 0.05 mg ml⁻¹ ascorbic acid (Sigma-Aldrich), 0.4 mM monothioglycerol (Sigma-Aldrich), 2 µM dorsomorphin (Peprotech), 10 µM SB-431542 (Miltenyi Biotec), 1% penicillin and streptomycin. On days 9–12, the medium was changed daily with DMEM/F12-based medium with B27 supplement, 0.05 mg ml⁻¹ ascorbic acid, 0.4 mM monothioglycerol, 20 ng ml⁻¹ BMP4 (Peprotech), 0.5 µM all-trans retinoic acid (Sigma-Aldrich), 3 µM CHIR99021, 1% penicillin and streptomycin. On days 12–20, the medium was changed every other day with DMEM/F12-based medium with B27 supplement, 0.05 mg ml⁻¹ ascorbic acid, 0.4 mM monothioglycerol, 10 ng ml⁻¹ FGF10 (Stemcell Technologies), 10 ng ml⁻¹ FGF7 (Peprotech), 3 µM CHIR99021, 50 nM dexamethasone (Sigma-Aldrich), 0.1 mM 8-bromoadenosine 3′,5′-cyclic monophosphate (Sigma-Aldrich), 0.1 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich), 1% penicillin and streptomycin.

hPSCs-derived MDLCs were dissociated with ReleSR solution for 8 min in 37 °C incubator, centrifuged, and resuspended at a concentration of 5 × 10⁶ cells/ml in DMEM/F12 medium (Gibco), mixed with Growth Factor Reduced Matrigel (Corning) at a ratio of 1:1 (vol:vol), dripped 20 μl/drop on the low adhesion plate (Corning), after gelatinisation at 37 °C for 30 min, added endometrium expansion medium (ExM)^{31 (link)} and gently scraped drops to 3D suspension culture. After 1 week, cell aggregates spheres were primed stimulated with 10 nM E2 (β-oestradiol, Sigma) in ExM for an additional of 1 week. Then the medium was replaced with 10 nM E2 + 1 μM P4 (progesterone, Sigma) + 1 μM cAMP (8-bromoadenosine 3’,5’-cyclic monophosphate, Sigma) in ExM and cultured for another 2 weeks in order to induced organoid maturation. The culture was continued for another two cycles (1 cycle: ExM for 1 week + ExM + E2 for 1 week + ExM + E2 + P4 + cAMP for 2 weeks) to evaluate organoids in long-term culture. The cell source of primary endometrial cells-derived organoids was from the non-pathological part of endometrium from hysterectomy due to leiomyoma in patient, which was approved by the ethical committee of the first affiliated hospital, school of medicine, Zhejiang university (ethics approval No. 2018-113)^{42 (link)}.

Acetonitrile, methanol, n-butanol, and ethyl acetate were of the HPLC grade, and guanine, adenine, inosine, guanosine, and 8-bromoadenosine-3′,5′-cyclic monophosphate standards applied in the analysis of nucleosides; nucleotides were purchased from Sigma-Aldrich (St. Louis, MO, USA).