Adipogenic and Osteogenic Differentiation of ADSCs

ADSCs abilities to differentiate into osteogenic and adipogenic directions were tested in vitro using standard differentiation and analysis protocols [11 (link)] in a normoxic environment. Briefly, osteogenic differentiation was induced by plating 6×10⁴ of ADSCs onto a 24-well plate and incubating in AdvanceSTEM Mesenchymal Stem Cell Media containing 10 % AdvanceSTEM Supplement (HyClone), 1 % antibiotic–antimycotic solution (HyClone), containing 10⁻⁸ М dexamethasone, 10 mМ β-glycerol-2-phosphate, 0.2 mМ 2-phospho-L-ascorbic acid (Invitrogen, USA) for 21 days. Differentiation efficiency was analyzed using Alizarin Red S staining for calcium accumulation. Adipogenic differentiation was induced by three cycles of consecutive incubation for six days in growth medium containing 10⁻⁶ М dexamethasone, 10 μM insulin, 200 μM indomethacin and 0.5 mM 3-isobutyl-1-methylxanthine (Invitrogen, USA) followed by three days incubation in growth medium containing 10 μM insulin. Cells accumulated intracellular lipids which were analyzed using Oil-Red-O staining.