In vitro analysis of the stemness properties in sorted CD133high
LC-42 cells. A. Cell sphere-forming assay under serum-free condition. 10% highest CD133+ cells and the lowest 10% CD133− were isolated via FACS. Spheres were generated from both sorted CD133high and CD133dim LC-42 cell populations within 3 weeks. Representative cell sphere images were acquired at 40× magnification. B. Colony-forming ability of different sorted LC-42 cell populations at different cell seeding numbers using soft agar clonogenic assay. The colony numbers of each cell population were compared at different cell seeding numbers. A direct relation between the number of cells seeded and number of colonies formed was observed. The clonogenic ability of different sorted cell populations showed no significant difference. The values of colony numbers of each cell population represented the average of three individual analysis. C. Recapitulate phenotype of the original LC-42 cells by post-sorting cell culture. FACS analysis of CD133 just following sorting (column a) and at the third week after sorting under the conventional cell culture condition (column b) showed the expressions of the CD133 in both CD133high and CD133dim cell fractions reached the similar level as its original LC-42 cells at the third week after sorting. D. Relative quantitative real-time PCR analysis of the expression of stem cell related genes Nanog, Oct4 and Sox2 in sorted CD133high and CD133dim LC-42 cell fractions. Real-time PCR assay revealed similar levels of these genes expression in CD133high and CD133dim cell fractions. The error bar reflected the variation within the triplicates.