Isolation and Culture of Mouse Adipose Stem Cells

ASCs were isolated from mouse subcutaneous, abdominal, and perivascular adipose tissue, as described previously (Gu et al., 2019 (link)). Briefly, adipose tissue was cut into 1-mm³ small pieces, which was then digested with 2 mg/mL collagenase type I (Gibco) and 1 mg/mL Dispase II (Sigma) in Hanks’ balanced salt solution (HBSS) at 37°C for 30–45 min with occasional vortex. The digestion was stopped by DMEM/F12 with 10% FBS and subsequently passed through a 100-μm filter followed by a centrifugation at 300 g for 5 min. Cell precipitates were resuspended in a stem cell culture medium as the following: Minimal Essential Medium-α (Gibco) with 15% fetal bovine serum (Gibco), 10 ng/mL recombinant human leukemia inhibitory factor (Sigma), 5 ng/mL recombinant human FGF (R&D), 2 mmol/L L-glutamine (Sigma), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco); they were then incubated in a 5% CO₂ incubator. The cells were passaged at a ratio of 1:3 every 2 or 3 days. The medium was refreshed every 2 days.