A, Phase-contrast image of passage 1–3 neurospheres formed by SFM1022 cells and WT-iPSCs. B, Immunostaining for Sox2 (green) and nestin (red). C, Immunostaining for GM2 (green). Blue represents DAPI staining. Scale bar indicates 200 µm (A), 50 µm (B) and 20 µm (C). D, The percentages of neurospheres formed by WT-iPSCs and SFM1022 cells, which expressed both Sox2 and nestin were determined. Data are the means ± S.E. and were obtained for five independent experiments. The number (E) and sizes (F) of passage 3–4 neurospheres of larger than 100 µm were determined. Data were analyzed using the Mann–Whitney U test and are shown as box-and-whisker plots. Boxes, 75th percentile with the median indicated; bars, 10th and 90th percentiles. Data were obtained for five independent experiments. n.s.: Difference not significant (P>0.05).