Induction of microRNA resistance and secretion in differentiating human endometrial stromal cells
نویسندگان
چکیده
Endometrial stromal cell culture Endometrial biopsies were obtained by curettage from consenting women aged 18-40 in accordance with local ethics committee requirements. Endometrial stromal cells were isolated and cultured as described previously (1). All experiments were performed within the first three cell passages. To induce decidualization in vitro, hESCs were treated with 0.5 mM 8-Bromo-cAMP 1 µM medroxyprogesterone acetate (MPA) (Sigma) in DMEM/F12 medium supplemented with 2% dextran-coated charcoal (DCC) treated fetal bovine serum (FBS), L-glutamine and antibiotic-antimycotic (Invitrogen, Paisley, UK). Where hESCs were treated in serum-free medium, all supplements, excluding DCC-FBS, were added to the medium. Vehicle treatments consisted of absolute ethanol at an equivalent concentration. Culture medium was normally replenished every two days. Cell line culture BeWo and Ishikawa cells were maintained in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS), L-glutamine and antibiotic-antimycotic (Invitrogen) HUVECs were maintained in Ham's F12 medium (Invitrogen) supplemented with 10% FBS, sodium bicarbonate, L-glutamine, antibiotic-antimycotic, heparin and endothelial cell growth supplement (Sigma). Human oocytes and embryos Women underwent ovarian stimulation and oocytes were collected by transvaginal ultrasound-guided aspiration and inseminated with prepared sperm (day 0). Oocytes were examined 19-20 hours after insemination, and classified as normally fertilised if two pronuclei were present. Fertilised embryos were cultured in MediCult media (Origio Ltd, Reigate, UK) to day 5 of development. Following embryo transfer, surplus embryos were donated to research by couples who gave informed consent. Eleven human embryos were used in this study, three at the blastocyst stage and 8 that had arrested development by day 5. Three of the embryos had holes made in the zona
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