FIG. 3.

Characterization of BMSCs. (A) Marrow cell cultures (top) and antibody-enriched BMSCs from 18-mo-old mice (bottom) were immuno-labeled with FITC-conjugated monoclonal antibodies as indicated and detected using a fluorescence microscope. (B) FACS analysis of enriched BMSCs. BMSCs from indicated ages of mice were labeled with FITC-conjugated monoclonal antibodies (CD45, CD11b, and Sca-1, respectively) and analyzed. Percentages of cells positive for CD45, CD11b, and Sca-1 are shown. This experiment was performed in triplicate. (C) Multipotentiality of enriched BMSCs. BMSCs isolated from 18-mo-old mice were exposed to osteogenic, adipogenic, and myogenic differentiation media. (a) Cells were treated with osteogenic supplements for 21 days and stained with ARS for mineralized bone nodules. (b) Cells were treated with adipogenic induction media for 2 days, cultured in maintenance media for 14 days, and stained with Oil Red O. (c) Cells were treated with 5-azacytidine (5-Aza) for 24 h, cultured in regular growth media for 21 days, and immuno-labeled with indicated antibodies. These experiments were performed a minimum of three times in triplicate. (d) Bone tissue labeled with GFP antibody to show osteoblast-like lining cells (arrows) 6 wk after injection of GFP-BMSC into mouse tibias. Star indicates GFP-BMSC embedded in the bone. These experiments were repeated at least three times with similar results.