A new approach to screening cancer stem cells from the U251 human glioma cell line based on cell growth state.

Cancer stem cells (CSCs) play important roles in the biological behaviour of malignant tumours. To study their properties, they must be carefully identified and purified. Cancer cells can acquire three different morphological types during single cell cloning. A small subpopulation of clones acquires a regular and compact shape, and these clones are enriched for CSCs; however, the majority of clones have an irregular morphology with loose intercellular junctions, with fewer characteristics of CSCs. At present, the main method to isolate CSCs is to collect the regular clones in low-density culture conditions; therefore, an insufficient amount of CSCs is obtained for clonal expansion. To obtain a more sufficient amount of CSCs, the clones with an irregular and loose morphology were examined in our study. We found a small subpopulation of U251 glioma cells that arrested in the suspended state and that subsequently migrated to form new clones. The suspended cells were isolated from the irregular and loose clones. Clonogenic assays were performed in which 43.70% of the suspended cells and 32.91% of the adherent cells formed new clones. To determine the biological differences between the suspended and adherent cells, carboxyfluorescein succinimidyl ester (CFSE) labelling, MTT assays, and cell cycle assays were performed. The results demonstrated that the suspended cells had the characteristics of CSCs, including higher proliferation rates, as well as self-maintenance and self-renewal capabilities, and they stained positively for markers of brain CSCs and had multilineage potential. Thus, we established a new and efficient approach for screening CSCs from the U251 human glioma cell line based on the cell growth state.