Primary spheres (A) and propagated spheres (B, C) show similar morphology and growth characteristics. (D) Analysis of the size (maximum diameter), the cell number, and the volume of primary, secondary, and tertiary spheres during a 7-day culture period using the neurosphere assay. After DIV 5 tertiary spheres showed a reduced ability to attach to coated culture dishes, which was required for cell counting. The sphere cell number was therefore only analyzed until DIV 5. Data are the mean±SD from three independent experiments. Scale bar=100 μm.