Tri-Lineage Differentiation of Mesenchymal Stem Cells

The differentiation capacity of MSCs was conducted using the Tri-lineage Differentiation Kit (Gibco BRL, Grand Island, NY, USA) as described previously [32 (link)]. For osteogenic differentiation, MSCs were seeded into 24-well plates and cultured with an osteogenic differentiation medium (Gibco) for 21 days. The osteogenic differentiation was confirmed by the appearance of Alizarin Red stain. For adipogenic differentiation, MSCs were cultured in an adipogenic differentiation medium (Gibco) for 7 days. The adipogenic differentiation was confirmed by the cellular accumulation of neutral lipid vacuoles, which were stained red with Oil Red O. For chondrogenic differentiation, MSCs were collected in 15-mL centrifuge tubes and cultured in a chondrogenic differentiation medium (Gibco). After 21 days of differentiation induction, the pellets were sectioned, and then, sulfated proteoglycans were visualized by staining with 1% toluidine blue (Merck, Darmstadt, Germany) for 10 min. The chondrogenic differentiation was confirmed by the appearance of Alcian Blue stain.