Figure 5
Ras-transformed astrocytes have an ability to form neurosphere-like colonies with the increased expression of CD133 mRNA. A, Expression of CD133 mRNA in NHA/TSE2 and NHA/TSR cells. The expression levels of CD133 mRNA were examined by semi-quantitative RT-PCR analysis. The number of PCR cycle was 32 for CD133. GAPDH is an internal control. 5Aza-dC; 5-Aza-2-deoxycitidine. TSA; Trichostatine A. B, In vivo tumor formation analysis. 5 × 106 of each cells were subcutaneously injected into NOD/SCID mice. Tumor formation was evaluated after 6 weeks of inoculation. The number of mice developing tumors/numbers of mice injected is shown. C, Representative images of NHA/TS and NHA/TSR cells cultured in neural stem cell medium. Bar graph shows mean ± s.d. of the absolute number of primary and secondary TSR spheres, which was measured from three independent experiments. Cell clusters ≥ 50 μm in diameter were counted as spheres. Secondary sphere assay was also performed at the same condition. Scale bar = 200 μm. D, Representative images of a sphere derived from single NHA/TSR cell. FACS-purified single NHA/TSR cell was cultured in NSC medium in a 48-well plate for 2 weeks. E, Individual TSR-derived clonal sphere was dissociated and reseeded in NSC medium (left) and in 10%FBS-DMEM (right) to confirm the secondary sphere formation activity and differentiation potential, respectively. Scale bar = 200 μm.