Figure 3
Neurospheres formed in 1%O2 presented with features of GSCs. (A–C) Single GL261 and U87 CD133−CD15−NESTIN− cell was seeded in the wells of 96-well plates and cultured in 1%O2, 21%O2 and 95%O2. Approximately 75% of cells survived after exposure to different oxygen levels for 3 days without a significant difference (d3 viable cells/d0 seeding cells). The neurospheres began to form after 1%O2 exposure for 3 d, and approximately 20% of cells (d7 spheres/d3 surviving cells) from GL261 and U87 formed neurospheres on day 7. The neurosphere formation rates then significantly increased, and 50.2% ± 4.167 from GL261 and 53.0% ± 3.391 from U87 (d14 spheres/d3 surviving cells) formed neurospheres after hypoxia exposure for 14 d. More surprisingly, the sphere rates eventually reached 93.1% ± 5.541 from GL261 and 95.6% ± 2.665 from U87 (d21 spheres/d3 surviving cells) under hypoxic conditions for 21 days. However, in normoxia or hyperoxia, no distinct neurospheres formed, and most cells remained a single cell and eventually died (* P < 0.05; ** P < 0.05; # P > 0.05, One-way ANOVA). (D,E) Newly formed GL261 neurospheres highly expressed the stem cell markers CD133, CD15 and NESTIN and the chemoresistance-related proteins ABCG2 and MGMT (* P < 0.05, Paired-samples T Test). (F) Trypan blue indicated there were no cells with blue-staining in the cytoplasm. (G) Asymmetric division was detected for newly formed neurospheres, and the results indicated cells in neurospheres maintained growth in suspension with a sphere morphology when cultivated in GSC medium for 5 d; however, adherent growth with a differentiation style was induced after 10%FBS administration.
