Isolation of stem-like cells from human MG-63 osteosarcoma cells using limiting dilution in combination with vincristine selection.

To isolate stem-like cells from the human MG-63 osteosarcoma cell line, different subpopulations of MG-63 cells were cloned by limiting dilution and passaged to obtain different sublines. The subline with highest clonogenicity was identified using a proliferation assay, cell-cycle analysis, and soft-agar colony-forming assay. The sublines were further selected in serum-free medium containing 20 ng/ml vincristine to identify cells that could form suspended sarcospheres. Identified cells were then characterized based on morphology, cell surface markers, adipogenic and osteogenic differentiation, and tumorigenicity in nude mice. A total of 19 holoclones that could be stably passaged were obtained. Sublines A1, A3, and D1 were markedly different from other sublines and the parental cell line. Subline D1 not only had a higher colony-forming efficiency and formed larger colonies, but also possessed a shorter latency of tumorigenesis in vivo. After subline D1 was cultured in suspension in medium containing vincristine, a highly enriched subpopulation of cells that could form sarcospheres and be stably passaged were obtained. These cells, designated as MG-63-M expressed multiple markers of multipotent or embryonic stem cells and possessed the capacity for self-renewal, multilineage differentiation, and significant multi-drug resistance. Thus, our results suggest that a subpopulation of stem-like cells can be isolated from human MG-63 osteosarcoma cell line.