Fig. 2

Neural differentiation of NPCs. A: Differentiated NPCs, at d21 after differentiation (upper row), stained for Oct4, Pax6, Map2, βIII-Tubulin, vGlut and vGat. Differentiated NPCs, at d0, d7, d14 and d21 after differentiation (lower row), stained for βIII-Tubulin. B: Quantification of fluorescence intensity of microscopy images of differentiated NPCs at d0, d7, d14 and d21 of differentiation, stained for Dcx, S100β, Map2 and βIII-Tubulin. C: Gene expression of neuronal (vGAT, vGLUT, BRN2, FOXG1), astrocyte (APOE, S100β) and oligodendrocyte marker (OLIG2) in differentiated NPCs at 7, 14 and 21d after neural differentiation, measured by qPCR. Total human brain RNA served as control. D: Experimental setup for determining the activity of differentiated NPCs and AAV construct for the expression of GCaMP6s and mRuby under the human synapsin promoter. E: Human synapsin promoter-based expression of mRuby in NPCs and differentiated neural cells. Confocal images of NPCs transduced with the AAV-mRuby construct (left), differentiated NPCs at d21 after differentiation (middle) and differentiated NPCs at d21 after differentiation and transduced with AAV-mRuby construct (right). F: Quantification of mRuby signal in transduced NPCs (left), non-transduced differentiated neural cells (middle) and transduced neural cells (right) from Fig. 1G. G: Confocal time-lapse images of non-transduced, differentiated neural cells (upper row) and neural cells transduced with AAV-GCaMP6s construct (middle row). After 350 s, cells were treated with 60 mM KCL (lower row). Scale bars: 50 µm. H: Quantification of GCaMP6s signal intensity from confocal time-lapse images. vGAT Vesicular GABA Transporter, vGLUT vesicular glutamate transporter, Dcx doublecortin, βIIITub βIII-Tubulin, hSyn human synapsin. Boxplots indicate the 25–75% quartiles of the data. Each dot in the plots represents one cell culture well and significance of mean differences between the groups was assessed using Tukey’s HSD. Line graphs are plotted as mean ± sem. Asterisks indicate significance: * p < 0.05, ** p < 0.01, *** p < 0.001