Isolation and Culture of AMSCs and BMSCs

AMSCs and BMSCs were isolated and cultured according to our previous study [26 (link)]. Briefly, AMSCs from the inguinal adipose tissue were collected and digested by type I collagenase (0.2%, Sigma, USA), filtered by 200-mesh sieve, centrifuged at 350×g for 5 min, resuspended in DME/F12 complete medium containing 15% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin/streptomycin/amphotericin B (Cellmaxin plus, Gendepot, USA), and plated onto 10-cm cell culture dishes at 37 °C with 5% CO₂. BMSCs were isolated from bone marrow of the femora which was flushed out with the DME/F12 complete medium. After repetitively pipetting, the bone marrow was plated onto 10-cm cell culture dishes at 37 °C with 5% CO₂. The medium was changed every 2 days and the cells (passage 0, P0) were subcultured when 80–90% confluence was reached. The P3 cells were used in the experiment.
In hypoxic condition described in our previous study [26 (link)], MSCs were cultured in DME/F12 complete medium for 7 consecutive days in a tri-gas incubator maintained 1% O₂, 5% CO₂, and 94% N₂ (MCO-5M, SANYO, Japan). Whereas in Tgf-β1 induction, MSCs were cultured in complete medium containing 10 ng/ml Tgf-β1 (Sigma, USA) for 7 consecutive days. The complete medium was changed every 2 days.