Figure 1
BMMSC and CPSC isolation and stem cell characterization. (A) Bone marrow aspiration procedure from a proximal humeral needle insertion site. (B) Shoulder joint replacement surgery with the humeral head collected for the biorepository bank. (C) BMMSC in a culture plate after their isolation. (D) CPSC isolated from joint cartilage and cultured in vitro in fibronectin-coated plates. (E) Cell proliferation analysis of in vitro cultured BMMSC and CPSC, expressed as doubling times at different passages (from P2 to P10). (F) Colony forming efficiency of BMMSC or CPSC collected at P2 and plated for 14 days at different cell numbers (50, 100, 200 and 500) (G) Cell proliferation, evaluated by AlamarBlue assay, of BMMSC and CPSC at different time points. (H) Cytotoxicity of CPSC compared to BMMSC in culture for 72 h. (I) Representative images of alcian blue glycosaminoglycan staining of chondrogenesis induced BMMSC and CPSC at 21 days. (J) Relative gene expression of chondrogenesis markers ACAN, SOX9 and COL1A1 at 0, 7, 14 and 21 days in induced cells compared to their control BMMSC (blue) and CPSC (red). (K) Representative images of von Kossa mineral deposition staining of osteogenesis induced BMMSC and CPSC at 21 days. (L) Relative gene expression of osteogenesis markers BGLAP, SPP1 and ALP at 0, 7, 14 and 21 days in induced cells compared to their control BMMSC (blue) and CPSC (red). (M) Representative images of Oil red O lipid deposits stained in adipogenesis induced BMMSC and CPSC at 21 days. (N) Relative gene expression of adipogenesis markers LEP, LPL and ADIPOQ at 0, 7, 14 and 21 days in induced cells compared to their control BMMSC (blue) and CPSC (red). (O) Flow cytometry percentages of negative anti-hematopoietic lineage cocktail (Neg. cocktail−) and triple positive anti CD90, CD105 and CD73 through different passages. Scale bar represents 200 μm. Asterisk represents statistically different means between groups (n = 3), *p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001.
