Fig. 5. Reduced osteoblastogenesis and increased adipogenesis of OG2-NcadC bone marrow cells. (A) Bone marrow stromal cells (BMSC) isolated from 2-month-old mice were cultured in 96-well plates in the presence of AA (50 µg/ml) and ß-GP (10 mM) for 4 weeks at low density (3.2x10³ cell/cm²) for limiting dilution analysis of CFU-Osteoblast, determined by Von Kossa staining. Relative to wild-type cells (WT), there was a lower number of von Kassa-positive wells in BMSC cultures isolated from OG2-NcadC (Tg) mice. (B) BMSC from OG2-NcadC and wild-type mice were cultured in the presence of an adipogenic cocktail (0.5 mM IBMX, 60 µM indomethacin, and 0.5 µM hydrocortisone) for 2 weeks and the number of CFU-Adipocyte was determined by Oil red O staining. A higher abundance of lipid droplet-positive cells was detectable even in cultures seeded at high density in cells from transgenic mice (10⁵ cells/cm²). Representative photomicrographs of wild-type and OG2-NcadC BMSC cultures stained with Oil Red O are shown on the right. (C) The number of CFU-Osteoblast was significantly lower, and the number of CFU-Adipocyte was significantly higher in transgenic compared with wild-type BMSC cultures, assessed by limiting dilution analysis (n=5); *P<0.05, t-test. (D) The abundance of PPAR-2, adipsin and Cbfa1 mRNA was assessed by real-time RT-PCR in BMSC cultures after 2 weeks of culture in the absence of stimulators. There was a significant increase in PPAR-2 and adipsin mRNA abundance in cells derived from OG2-NcadC animals compared with wild-type littermates, whereas Cbfa1 mRNA was reduced 40%. (E) Expression of the transgene, NcadC was detected after 2 weeks of culture by RT-PCR of total RNA extracted from parallel cultures, with GAPDH as control for RNA integrity and abundance (lower panels). No PCR products were obtained in the absence of reverse transcriptase (RT), thus excluding DNA contamination.