Figure 3
Notch inhibition disturbs the maintenance of stem cell characteristics of neuroprogenitor cells in the hESC-derived neuroectodermal spheres. (A) Disappearance of the rosette structure with DAPT treatment. Neuroectodermal spheres (NESs) derived from CHA-hES3 were cultured with or without DAPT (5 μM) for 4 days. Rosette-like folded structures in NESs were markedly reduced with DAPT treatment. CHA3 hESCs were used. Scale bars, 500 μm. (B) DAPT-induced loss of rosette structures. DAPT treatment removes rosettes from attached NES clumps whereas the DMSO control group still contains rosettes. H9 hESCs were used. Scale bars, 200 μm. (C) Reduction in volume of the DAPT-treated NESs. The volume of NESs after DAPT treatment for 4 days was measured. Control group (black) was estimated as 0.066 ± 0.042 mm3 (mean ± standard deviation (SD); n = 44) and the DAPT group (red) was 0.039 ± 0.027 mm3 (n = 58). The difference between the two groups was significant (p < 0.001). In the box plot, box percentage and mean values (open squares) are indicated. Whiskers range from 5–95%. 'x' in each box indicates an upper and lower extreme value. (D) RT-PCR results. Neural stem cell marker genes (SOX1, VIM, and NES) and Notch-regulated target genes (HES1 and HES5) showed reduced expression levels in DAPT-treated NESs derived from CHA3 and H9 hESCs. However, expression of the pro-neuronal genes, NGN1 and MASH1, were reversely increased by DAPT-treatment. (E) Neuroectodermal sphere-reforming assay. NESs were dissociated into single cells and cultured for 17–18 days with (gray) or without (black) DAPT (5 μM). Among the newly formed NESs, those larger than 50 μm diameter were counted. The values denote mean ± standard error and there are significant differences between the control and the DAPT-treated groups (p < 0.05). Experiments were independently performed three times. (F) BrdU incorporation assay. The proportion of mitotically active cells was reduced in DAPT-treated NESs. The statistical values denote mean ± standard error, and 'n' indicates the number of cells counted. Data were obtained from more than three (CHA3, p < 0.05) or five (H9, p < 0.005) independent experiments.