Figure 5.

In vivo differentiation of mc13 cells into osteogenic lineage after genetic engineering to express BMP-2. The amount of BMP-2 secreted by the mc13 cells that were transduced with adBMP-2 was found significantly higher (*P < 0.05) than the nontransduced mc13 cells (A). 0.5–1.0 × 10⁶ cells genetically engineered to express BMP-2 were injected into hind limbs of SCID mice. After 14 d, mice were killed, and the hind limb muscle tissues were analyzed radiographically for evidence of bone formation. There was a robust ectopic bone formation (seen radiographically) within skeletal muscle in all mice injected with mc13 cells transduced with adBMP-2 (B, arrow). The injected muscle containing the ectopic bone was then harvested and stained for β-galactosidase activity to locate injected cells. The LacZ positive cells were uniformly found within the lacunae, a location where osteoblasts and osteocytes are normally found (C). The ectopic bone was also stained for presence of dystrophin. As indicated by green fluorescence, the ectopic bone contained abundant cells expressing dystrophin, confirming that mc13 cells were active participants in formation of bone (D). To determine whether the genetically engineered mc13 expressing BMP-2 can express bone protein, we colocalized β-galactosidase expressing nuclei, osteocalcin expression, and nuclei staining (DAPI) by immunohistochemistry (E–H). We identified nuclei expressing β-galactosidase (see Fig. 6 F, arrow, FITC/green) that expressed osteocalcin (see Fig. 6 G, arrow, cy3/red) and colocalized with nuclei staining (see Fig. 6 E, arrow, DAPI/blue). The triple colocalization of DAPI/osteocalcin and β-galactosidase (Fig. 6 H, arrows) suggests that the genetically engineered mc13 can express bone protein (osteocalcin). We have also observed β-galactosidase expressing nuclei (Fig. 6 F, arrowhead) that were not colocalized with osteocalcin expressing cells (Fig. 6 G, arrowhead), suggesting that some of the engineered mc13 were not expressing osteocalcin (Fig. 6, E–H, arrowheads). Bar: (C and D) 50 μm; (E–H) 25 μm.