Evaluation of differentiation properties of SP cells. (A) In vitro chondrogenesis. SP and NSP cells were cultured for 21 days in TGF-β contained chondrocyte differentiation medium. (A-a) Pellets were stained with toluidine blue and alcian blue. (A-b) Expression of chondrogenic genes was also examined. SP and NSP cells were cultured for 21 days, and chondrocyte specific gene expressions were determined by RT-PCR analysis. Representative data were shown with 3 experiments. (B) In vitro osteogenesis. SP and NSP cells were cultured for 21 days in osteocyte differentiation medium. (B-a) ALP activity was determined in both derivatives. (B-b) Calcium deposition was examined by 1% Alizarin red staining, High-magnification image of stained cells derived from SP cells were shown in right panel. Scale bar is 200 μm. (B-c) Expression of osteocyte specific gene Osteocalcin was determined by RT-PCR. (C) In vitro myogenesis. SP and NSP cells were cultured for 7 days in myocyte differentiation medium. Desmin expressions were determined by immunocytochemical staining (C-a; scale bar = 100 μm), and high-magnification image of SP derived myocyte (C-b; scale bar = 50 μm). (C-c) Myocyte specific gene expressions were determined by RT-PCR.