Figure 4

Inhibition of Notch signaling directs cells of neuroectodermal spheres to differentiate into neuronal cells. (A) Immunostaining of attached NESs for TUJ1. NESs derived from H9 hESCs were treated with DMSO or DAPT for 4 days and stained for TUJ1 (green). Images of NESs are shown in parts in a and b or as whole clumps in c and d. In d, two consecutive pictures separately taken are combined. (B-C) Measurement of TUJ1-positive cells after DAPT treatment. NESs cultured in the NSM containing DMSO (solid bars) or DAPT (gray bars) for 4 days were dissociated into single cells and attached on the matrigel-coated coverslip before immunostaining for TUJ1 (B) and Nestin (C). The number of positively stained cells relative to DAPI-positive cells was calculated. The values denote mean ± standard deviation and there are significant differences between the control and the DAPT-treated groups (p < 0.005 for TUJ1 and p < 0.001 for Nestin). Experiments were independently performed five times and 2087 cells in total were counted. Results of Western-blot analyses of DMSO control and DAPT treated samples are shown in the boxes, in agreement with the results of cell counting. TUJ1 and MAP2 are neuronal markers, and S100, GFAP, NG2 and CNPase are glial markers. α-Tubulin, a control for the bands in B and C. (D) Enrichment of neurite bundles by withdrawal of growth factors. The rosettes structures are clear, regardless of GF presence (arrowheads) but not visible in the DAPT-treated samples. Neurite formation was markedly accelerated in the DAPT-treated NESs after withdrawal of growth factors such as bFGF, LIF and EGF (arrows). Such neurite bundles are rarely seen in DAPT-treated NESs in the presence of growth factors. Scale bars, 200 μm in A and 500 μm in D.