Abstract： Objective To investigate morphological and differential heterogeneities of U251 glioma cell colonies,and identifying the colonies comprised of tumor stem cells.Method Colony formation assay was employed to observe and classify the colonies derived from U25 glioma cells,then glial fibrillary acidic protein(GFAP)and nestin were labeled in all colonies to determine differentiation status.Furthermore,we isolated and cultured the colonies with the poorest differentiation,and the properties of tumor stem cells:self-renewal,multiple lineage differentiation potential,and expression of stem cell marker CD133 were assayed.Results Colonies derived from U251 glioma cells had three types:tight,transitional and loose colonies.Among these colonies,the tight colonies showed the poorest differentiation.U251 cells of tight colonies possessed the properties of self-renewal and multiple lineage differentiation.Moreover,they were found to be able to express CD133 and nestin.Conclusions U251 cell colonies varied in morphology and differentiation,and the tight colonies might he comprised of tumor stem cells.Our study provided a convenient and efficient method for cloning glioma stem cells.