Tri-lineage Differentiation of MSCs

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).