Isolation and Culture of Mesenchymal Stem Cells

MSCs were isolated as previously described [19 (link)]. Briefly, cells were isolated from patients undergoing hysterectomy at the Johns Hopkins University Hospital. The Institutional Review Board of the Johns Hopkins University reviewed and approved the study (IRB00196175), and informed consent was obtained from all patients to participate. Tissue was transferred to the laboratory immediately after surgery and washed several times with a Hanks’ Balanced Salt Solution (HBSS, Thermo Fisher Scientific, Waltham, MA, USA) without calcium or magnesium. Tissue was then manually cut into 1–2 mm³ specimens and incubated in sterile HBSS (without calcium, or magnesium) with collagenase (Worthington, Lakewood, NJ, USA), deoxyribonuclease (DNase, Sigma-Aldrich, St. Louis, MO, USA), antibiotic–antimycotic mixture (Thermo Fisher Scientific), and HEPES buffer solution (Thermo Fisher Scientific) at 37 °C on a shaker for 4–8 h. The digest was filtered through a 100 µm and then a 40 µm filter and cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F12 (DMEM/F-12) (Thermo Fisher Scientific) medium supplemented with HEPES, L-glutamine, 10% FBS, and 1% antibiotic–antimycotic.