Characterization Of Side Population And Other Stem Cell Phenotypes In U251 Cells Under Stem Cell Culture

Objective:1,To investigate the utility of side population assay in glioma stem cells research;2,To analyze the phenotypic characteristic of the U251 cells under stem cell culture;3|,To verify methodological approach to sort and target glioma stem cells.Methods:we transfered the adherent U251 cell line routinely cultured in the serum-containing into serum-free media, supplemented with EGF and b-FGF, and assaied the propotion of SP cells using flow cytometry ; Meanwhile, other phenotypes of glioma stem cells including immunophenotype CD133 (flow cytometry, immunofluorescence assay), Nestin expression(immunofluorescence assay), self-renewal capability (limiting dilution assay), and in vivo tumorigenicity (xenograft immunodeficient mice) were simultaneously evaluated.Results:1,The proportion of the SP cells in U251 cells adherently grown in serum-containing media was 0.54%±0.39%, when cultured for three months in serum-free media ,the propotion rose to 5.51%±2.32%(P<0.05); 2,There were no CD133(+) cells in both the adherent cells in serum-containing media and the neurosphere cultured in serum-free media;3,There were 8.17±3.74 Nestin (+) cells per high magnification field in the U251 adherent cells under serum-containing media,but the percentage rose to 37.39±6.31 when transfering the cells to serum-free media(P<0.05);4,Comparing the self-renewal capacity of the adherent cells and the neurospheres, there was significant difference using limiting dilution assay(P<0.05);5,The tumor-forming ratio in U251 adherent cells group was 6/15, while the ratio was 12/15 in tumor neurospheres implanting group; and the occurrence of neurological symptoms in tumor spheres implanting group was earlier than the adherent cells group.Conclusions:1,The proportion of the SP cells in U251 adherent cells under serum-containing media was extremely low,but when cultured for three months in serum-free media ,the propotion increased significantly;2,Together with the increase of the SP propotion, other"stemness"characteristics such as nestin expression, self-renewal capacity , and the tumorigenicity in vivo were enriched correspondingly;3,The changes of the"stemness"characteristics in stem cells abovementioned are independent of the expression of CD133;4,It was unclear whether the increased propotion of SP cells and corresponding"stemness"characteristics were due to the enrichment of originally existing GSC in the U251 cell line, or due to a retro-differentiation process occuring in vitro condition, which remains to be answered.