Characterization and Multilineage Differentiation of GMSCs

Characterization of colony forming unit-fibroblasts (CFU-Fs) and multi-differentiation capacity of GMSCs were performed as per previously described methods [28 (link), 31 (link)]. Isolated mononuclear cells were seeded at a density of 1 × 10³ per dish in 100-mm culture dishes containing the regular medium. Adherent cells were cultured for 18 days. The cultures were subsequently treated with PBS containing 0.1% Crystal violet and 4% paraformaldehyde (PFA). Cell clusters containing > 50 cells were counted as single colonies (CFU-F) under a Primovert microscope (Zeiss). GMSCs at the 3rd-5th passages were used for multi-differentiation capacity into adipocytes, osteoblasts, and chondrocytes. For adipogenic differentiation, GMSCs at 70%–80% confluence were cultured in an osteogenic medium containing α-MEM supplemented with 0.25 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 5 μg/mL insulin (Cell Science & Technology Institute, Sendai, Japan), and 2 mM glutamine. For osteogenic differentiation, GMSCs at 70%–80% confluence were cultured in an osteogenic medium containing α-MEM supplemented with 2 mM β-glycerophosphate (Wako, Osaka, Japan) and 50 μg/mL ascorbic acid (Wako). For chondrogenic differentiation, GMSCs (5 × 10⁵ cells) were cultured in MesenCult™ -ACF, a chondrogenic differentiation medium (STEMCELL™ Technologies, Canada), according to the manufacturer’s instructions.