Background & objective: Tumor stem cells (TSCs) possess the ability of chemoresistance. This study was to isolate and identify TSCs from human glioma cell line U87.
Methods: After U87 cells were grown as monolayer attached at the bottom of flasks in the medium containing serum, neural stem cell culture medium containing 5 ng/ml vincristine was added to obtain the first generation of tumorosphere. Then, the first generation of tumorosphere was dissociated into single cell suspension and seeded in the neural stem cell culture medium to obtain the second generation of tumorosphere. The morphology of the first and the second generations of tumorosphere was observed by light and scanning electron microscopy, respectively. The expression of nestin, glial fibrillary acidic protein (GFAP), beta-tubulin III and myelin basic protein (MBP) in the second generation of tumorosphere and differentiated cells were determined by immunofluorescence assay and confocal laser scanning microscopy.
Results: The first and second generations of tumorosphere were obtained successfully. Cells inside the second generation of tumorosphere with a few protrusions attached with each other tightly. The expression of nestin, a stem cell marker, was detected in the second generation of tumorosphere. The expression of nestin, GFAP, beta-tubulin III, MBP was detected in the differentiated cells derived from the second generation of tumorosphere.
Conclusion: Tumor stem cells with the capacity of self-renewal and multipotency exist in U87 cells. Selective culture of tumor cells with treatment of vincristine might be a simple and practical method for isolation of TSCs.