The effect of growth factor addition on neurosphere size, proliferation and expression of cell differentiation markers in eOBSC neurospheres.
The eOBSCs were cultured and passaged as floating neurospheres as described in Fig. 1. After 4 days in culture, BrdU (5 µM) was added for 30 minutes and the neurospheres were collected on matrigel, immunostained and counterstained with DAPI. Images show the representative neurosphere growth in cultures to which factors were added daily (A, D, G), every 2 days (B, E, H) and every 4 days (C, F, I), and that were immunostained with BrdU (A–C), GFAP (D–F) and TuJ1 (G–I). Bar graphs show the number of DAPI+ cells (J), and the percentages of BrdU+ (L), GFAP+ (M) and TuJ1+ (N) cells. Line graphs show the distribution of neurosphere number versus volume (K). Results represent the mean ± SEM from 15–30 neurospheres in 2 experiments. *P<0.05 (One way ANOVA followed by post hoc analysis using Bonferronís test). Scale bars (I) = 77.51 µm, insert = 39 µm.