Gliomas are the most common tumors of the central nervous system (CNS) and a frequent cause of death. The treatment of malignant gliomas is often palliative due to their high recurrence rate. A growing body of evidence suggests that glioma may arise from cancer stem cells (CSC) correlated with neural stem cells (NSC), with the capacity for self-renewal and multipotency. CSCs have been isolated from human gliomas and numerous other solid tumors. It is assumed that a number of established malignant cell lines also contain a rare subpopulation of stem cells. This study was designed to investigate the proportion of CSCs in the human glioma cell line SHG44 and to study the limitations of CD133 immunophenotyping in glioma stem cell research. SHG44 cells were cultured in both serum-containing and serum-free medium. The similar shape in growth curves (in the exponential growth phase) revealed that most cells participated in the population amplification. Time gradient BrdU labeling and monoclonal assay revealed that almost every single cell participated in the division growth (98.82%) and possessed the ability to form clones (96.19%). No significant difference was found in the proportions of CD133+ cells in the serum-containing and serum-free groups (38.25%/37.92%). In addition, no significant difference was noted in the proportions of CD133+ cells among monoclones selected randomly in the serum-containing group. These results suggested that CD133- cells generate CD133+ cells and have the ability to form clones. Thus, we concluded that most SHG44 cells were CSCs and serum-free medium was not necessary for the generation of CSCs. In this line, CD133- cells also possessed clonogenic, self-renewal capacities and were also CSCs.

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