Fig. 4. Simultaneous nestin expression and aggregates formation. After 72 hours incubation in serum-free medium, the cells were trypsinised and resuspended in DEM/F12 with N2 and B27 for 24 hours. During that time, passage 15 cells aggregated to form clusters or aggregates resembling neurospheres (A), whereas passage 4 rSCs remained in suspension (B). Nestin-negative passage 15 cells immediately adhere on the culture dish (C). The spheres obtained in condition A were then placed on polyornithine for 5 days in DEM/F12, N2 and B27 (E) where they exhibited a different morphology than that observed in DEM/20% FBS medium (D). Nestin immunocytochemistry (green) reveals that only 15% of the cells still express nestin under these conditions (F). Scale bar: (A-F) 40 µm.