Triple staining to identify cell fusion. Human marrow cells (5000) from a long-term culture were cocultured with C2C12 cells (5000) for 4 days under conditions that favored proliferation. The cultures were then switched to differentiation media for 2 days to induce myogenic fusion. Myotubes were revealed by sarcomeric myosin heavy chain monoclonal antibody MF20 (A). Human nuclei were detected by antibody specific to human nuclei (B). The fusion of human marrow cells (arrowhead) with myotubes was detected by correlating the human nuclei with nuclear spaces in the myotube (C). In contrast, unfused stromal cells (arrow) did not align with the myotube nuclear space. Total cell nuclei in the culture were stained with DAPI (D, blue). The same results were observed from CD45-depleted primary stromal cells and mesenchymal stem cells. However, human stromal cell lines HS-5 (E) and HS-27a (F) failed to fuse with C2C12 in coculture. Original magnifications: × 200 (A-D) and × 100 (E-F).