Differentiation and proliferation of mouse embryonic NSPCs induced by ephrin-A1 and/or FGF2 (A) NSPCs cultured as adherent cells.

<p>NSPCs were plated as adherent single cells at 1000 cells/well on a PLL- and fibronectin-coated cover glass in culture medium containing FGF2 (20 ng/ml) and EGF (20 ng/ml) for 3–5 days. Cells were then stained for anti-nestin (green), and counterstained with DAPI (blue) for nuclei. Scale bar: 50 μm. (B) NSPCs were plated as single cells 500 cells/well on PLL- and fibronectin-coated 96-well plates for 3–5 days in medium containing the different reagents. Reagents were re-added on days 3 and 5, and the numbers of Tuj1-positive cells counted between days 8 and 10 under an inverted fluorescent microscope with an optical filter. Scale bar: 50 μm. (C) The number of neuronally differentiated Tuj1-positive cells per 1000 cells. Values were analyzed using Tukey’s multiple comparison test following ANOVA (two-way). Bars represent the SD. * p<0.05, ** p<0.001 (n = 6) compared to the control. (D) The number of total cells in the area of one microscopic field. Values were analyzed using Tukey’s multiple comparison test following one-way ANOVA. Bars represent the SD. * p<0.05, ** p<0.001 (n = 6) compared to the control. (E) Effect of various signal inhibitors on the number of neuronally differentiated Tuj1-positive cells per 1000 cells (upper panel) and on the number of total cells in a single microscopic field (lower panel). NSPCs were plated as single adherent cells at 2 × 10⁵ cells/well on PLL- and fibronectin-coated 4-well chamber slides for 2 days in growth medium. Medium was replaced with growth factor-free medium containing ephrin-A1 and various inhibitors on day 2, and the number of Tuj1-positive cells and total cells per unit area counted on day 4 under an inverted fluorescent microscope with an optical filter. SU (SU5402), FGFR inhibitor; LY (LY294002), PI-3 kinase inhibitor; U (U0126), MEK1 and MEK2 inhibitor. Bars represent the SD. Values were analyzed using Tukey’s multiple comparison test following one-way ANOVA. * p<0.05, ** p<0.005, *** p<0.0001 (n>8) compared to the control (IgG(Fc)).