Tri lineage differentiation kit

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.

MSC differentiation was conducted using the Tri-lineage Differentiation Kit (Gibco BRL, Grand Island, NY, USA) as described previously 43 (link). For adipogenic differentiation, MSCs seeded in a 24-well plate at a density of 8 × 10⁴ cells per well were cultured in the adipogenic differentiation medium for 21 days with the consumed medium refreshed every three days. Derived adipocytes were stained red with Oil Red O (Sigma, St Louis, USA). For osteogenic differentiation, 4 × 10⁴ MSCs seeded in a 24-well plate were cultured in the osteogenic differentiation medium for 21 days with the consumed medium refreshed every three days. Derived osteocytes were stained positive for Alizarin Red. For chondrogenic differentiation, 2 × 10⁵ MSCs seeded in a 24-well plate were cultured with the chondrogenic differentiation medium for 21 days with the consumed medium refreshed every three days. Chondrocytes were stained positive with 1% toluidine blue (Sigma, St Louis, USA).