Differentation media bulletkits osteogenic

To induce osteogenic differentiation, HASCs, harvested at passage #5 and with 60–70% confluency, were cultured in Differentation Media BulletKits-Osteogenic (Lonza), according to manufacturer’s instructions. The differentiation potential for osteogenesis was assessed by mineralization of calcium accumulation on von Kossa staining (Abcam, Bristol, UK), according to producer’s instructions; moreover, we determined changes in RT-PCR expression of specific genes, namely, Secreted Phospho-Protein 1, Bone Gamma-carboxyglutamate (Gla) Protein (BGLAP), Runt-related transcription factor 2 (RUNX2), Alkaline Phosphatase, Liver/bone/kidney (ALPL). To induce adipogenic differentiation, HASCs harvested as indicated above were cultured in Differentiation Media BulletKits-Adipogenic (Lonza). The potential for adipogenic differentiation was assessed by Sudan III staining (Sigma-Aldrich), according to the manufacturer’s instructions. Changes in the expression of specific genes, markers of adipogenic differentiation, such as Peroxisome Proliferator-Activated Receptor Gamma (PPARG), Lipo-Protein Lipase (LPL), Fatty Acid Binding Protein 4 (FABP4), were also determined by RT-PCR.