Figure 9. Decreased osteoblast differentiation in MSCs of MC3R^hDM/hDM mice.

MSCs were isolated from compact bone of tibia and femur from chow-fed female MC3R^hWT/hWT and MC3R^hDM/hDM mice at 7 weeks of age. Cells were isolated from one mouse for each group, and six independent experiments were performed (n=6/group). Isolated MSCs were cultured in 25-cm cell culture dishes for 4 passages (see methods for detailed information). MSCs were used (a) for western blotting to examine MC3R protein expression or (b–f) MSCs were differentiated into osteoblasts for 14 days to examine differentiation capacity. (b–c) Cultured MSCs differentiated into osteoblasts after Alizarin red S staining. (d–e) Microscopic images (10X) of osteoblasts after Alizarin red S staining. Scale bar, 100 μm. (f) Stained Alizarin red S was extracted from osteoblast and quantified at 450 nm. (g–j) Confocal microscopic images of osteoblasts stained with Alizarin red S (red) (g,h) or osteocalcin (red) (i,j). Nuclei were stained with DAPI (blue). Representative maximum intensity projection images are shown. Scale bar, 25 μm. (k) qPCR analysis of genes related to osteoblast differentiation (7 days after differentiation). Similar results were found for male mice (data not shown). Data are represented as mean±s.e.m. for f and k. Groups were compared by Student's t-tests (two-tailed) (f,k).