Figure 1. Endothelial cell contact enforces neural stem cell quiescence and promotes stem cell identity.
(a) Phase contrast and fluorescence images of cell-tracker labelled (green) SVZ cells cultured alone (−), in the presence of endothelial cell-secreted soluble factors (+Sol) and in direct co-culture with indicated endothelial cells (+bEND, +bmvEC). Cultures were imaged 24h after seeding. Merged images are shown for clear identification of the NPC in mixed cultures. (b) Quantification of PI-BrdU FACS profiles of cultures described in (a) following a 1 hour BrdU pulse. For this and all later experiments NPC were separated from endothelial cells prior to analysis (n=3 dishes from independent experiments for all bEND, n=2 for Sol bmvEC transwell and n=4 for bmvEC cocultures). Error bars denote s.e.m. One-way ANOVA with Bonferroni correction. (c) Quantitative RT-PCR analysis of mRNA levels of type-B and type-C marker genes in the indicated culture conditions expressed as fold change relative to control NPC monocultures (n=3 RNA extract from independent experiments). Error bars denote s.d. One-way ANOVA with Bonferroni correction. (d) Representative immunofluorescence images of NPC seeded alone (−) or in coculture with endothelial cells (+bEND, +bmvEC) for 24h and stained for the indicated type-B and type-C markers. All numbers embedded in the figures indicate the percentage of marker positive cells in each condition. (e) Representative FACS profiles and quantification of EGFR positive cells in the same cultures as in (d). (f) FACS plots and quantification of GFP+ NPCs isolated from Hes5-GFP reporter mice and cultured as in (d). For these and all later experiments for which n<5, individual data point are shown as dots. Source data in Supplementary table 1. For this and all later figures ***=p<0.001, **=p<0.01, *=p<0.05. Scale bars: a=30μm; d=75μm