Mouse and Human Adipose Cell Differentiation

Mouse mesenchymal cells (D1, ATCC CRL-12424) were grown and maintained routinely in standard medium containing low glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% L-glutamine and 1% non-essential amino acids. When cells reached 80% confluence, adipogenic differentiation was induced with adipogenic (‘AD’) medium prepared with standard medium supplemented with 1 μM dexamethasone (Cayman Chemicals, USA), 100 µM isobutylmethylxanthine (IBMX) (Sigma-Aldrich, UK), 1 μM rosiglitazone (Cayman Chemicals, USA), and 10 μg/ml insulin (Sigma-Aldrich, UK). All supplements were prepared according to the manufacturer’s guidelines. AD medium was used throughout the treatment period, as previously described^{55 (link),56 (link)}. Experimental cultures were treated for 9 days in two groups: cells differentiated under standard cell culture thermal conditions (37 °C), and cells differentiated at 32 °C^{57 (link)–59 (link)}. Matching undifferentiated control cells maintained in standard medium were also grown at 37 and 32 °C. The same protocol was applied to human adipose-derived stem cells (hADSCs) (ThermoFisher UK, Cat. No R7788-115) and primary mouse BM-derived stem cells. For the analyses performed in this study, 3 batches of cells were analysed.