Adipogenesis induction medium

Osteogenic and adipogenic differentiation was performed according to the manufacturer’s instructions (Differentiation Media BulletKits, Lonza, Basel, Switzerland). To induce osteogenesis, WJ-MSCs were seeded at 3.1 × 10³ cells/cm² in complete medium in a 12-well plate. After 24 h, Osteogenesis Induction Medium (Lonza) was added to the adherent cells; the medium was replaced twice per week, and differentiation was continued for 21 days. The MSC control consisted of cultivating the cells with only basal medium. At day 21, calcium mineralization was assessed by coloration with Alizarin Red (Sigma-Aldrich, Saint-Louis, MO, USA). For adipogenic differentiation, WJ-MSCs were seeded at 2.1 × 10⁴ cells/cm² in complete medium in a 12-well plate. At 100% confluence, three cycles of induction/maintenance were performed. Each cycle consisted of feeding MSCs with supplemented Adipogenesis Induction Medium (Lonza) and culturing for 3 days, followed by 1 to 3 days of culture in supplemented Adipogenic Maintenance Medium (Lonza). After three complete cycles, WJ-MSCs were incubated with Adipogenic Maintenance Medium for 21 days; the medium was replaced twice a week. The MSC control consisted of cultivating the cells with Adipogenic Maintenance Medium only. After 21 days, staining with Oil Red O solution (Sigma-Aldrich, Saint-Louis, MO, USA) was performed to detect lipid droplets.

Osteogenic, adipogenic, and endothelial differentiation of ASS1-HLSCs was performed as described previously [4 (link)]. Briefly, ASS1-HLSCs were cultured in osteogenesis induction medium (Lonza) for 3 weeks (replenished twice per week for 3 weeks). Differentiation was evaluated by fixing cells with 4% paraformaldehyde for 20 min and then staining with alizarin red, pH 4.1 (Sigma-Aldrich), at room temperature. For adipogenic differentiation, cells were cultured in adipogenesis induction medium (Lonza), followed by adipogenic maintenance medium (Lonza). To evaluate the differentiation, cells were fixed with 4% paraformaldehyde for 20 min at room temperature then stained with 0.5% oil red O (Sigma-Aldrich) in methanol (Sigma-Aldrich). Endothelial cell differentiation was obtained by culturing ASS1-HLSCs in EBM (Lonza) with vascular endothelial growth factor (10 ng/ml; Sigma-Aldrich) for 10 days. Following culture, endothelial differentiation was evaluated by flow cytometric analyses of endothelial markers as described previously [4 (link)].