Figure 5

D5E2-P1 expression corresponds with Msi1-positive NS/PCs and astrocytes. (A), (B) D5E2-P1 was active in primary neurospheres. (A) Primary neurospheres derived from ESC lines, on day 6. Scale bar: 100 μm. (B) Three cell lines with intense reporter expression were selected from 47 ES cell lines; EB(+RA) were selected and primary neurospheres were formed in each clone. The reporter activity was analysed on day 6. (C) Cells from secondary neurospheres were differentiated and stained with each antibody shown. D5E2-P1 showed intense GFP expression. GFP was not induced by P1, and was only slightly induced by D2E-P1. Vertical arrowheads show Marker(+)/GFP(+) cells. D5E2-P1 induced GFP expression in Msi1(+) and Nestin(+)/GFAP(+) cells. Scale bar: 50 um. (D) Immunofluorescence intensity was visualized by scatter plots. The vertical axis shows the intensity of Msi1 and each cell-type-specific marker. The horizontal axis shows GFP intensity. The ratio of marker(+) GFP(+)/GFP(+) (%) cells is indicated at the bottom of the boxes.