Fibroblast Growth Factor-2 Primes Human Mesenchymal Stem Cells for Enhanced Chondrogenesis
Figure 4
FGF-2 primes hMSCs for CG by increasing basal Sox9 protein levels.
(A) Human MSCs were exposed to FGF-2 for 30 minutes or 2 hours and analyzed for Sox9 protein levels prior to pellet culture by Western blot analysis. Exposure to 5 and 50 ng/mL FGF-2 led to increased Sox9 protein levels in a dose-dependent manner. GAPDH was used as a loading control. (B) Human MSCs were exposed to FGF-2 for 2 hours and chondrogenically-induced in pellet culture. FGF-2-expanded hMSCs (+) showed elevated pSox9 protein levels by Western blot analysis. GAPDH was used as a loading control. (C) Human MSCs were treated with or without SB431542 for 15 minutes, exposed to combinations of 5 ng/mL FGF-2, 10 ng/mL TGF-β1 and 10 µM SB431542 for 2 hours, and analyzed for Sox9 mRNA and protein levels by end-point PCR and Western blot analysis, respectively. Exposure to FGF-2 led to increased Sox9 mRNA and protein levels, while TGF-β1 reduced Sox9 expression. Smad7 gene and pSmad3 protein levels were analyzed to confirm successful knockdown of TGF-β signaling. GAPDH was used as a loading control. (D) Human MSCs were expanded in FGF-2 for varying durations of time and analyzed for Sox9 protein using flow cytometry. Sox9 protein levels per cell were elevated at all time points after FGF-2 exposure compared to the control and gradually increased with longer exposures. Values are represented as fluorescence signal to noise ratios using geometric means.
