Fig. 5.

Delayed neuronal differentiation of TSC NPCs. (A) Representative confocal images of DIV7, 14, and 21 neuronal cultures generated from TSC and control NPC lines. An increasing proportion of the cells express the neuron-specific markers HuC/D and MAP2 over the time course of differentiation. Nuclei are counterstained with DAPI. Scale bar = 100μm. (B, C) Automated high content analysis of randomized image fields shows a steadily increasing percentage of HuC/D+ cells during the time course of differentiation for TSC and control cultures in both subject sets. However, at DIV7 TSC cultures showed a significantly lower percentage of HuC/D+ cells than control in both subject sets (*p<0.05, n = 4 independent differentiation experiments). The percentage of HuC/D+ neurons in TSC cultures was not significantly altered at DIV14 or DIV21 in either subject set (n = 3–4 independent experiments). Significance at each time point was determined via multiple unpaired t tests corrected using the Holm-Sidak method and α= 0.05. (D) Western blotting of DIV7 cultures show reduced levels of early neuronal markers DCX and βIII-TUB in patient samples from both subject sets. (E, F) Quantification of Western blot data normalized to internal controls. The decrease in DCX and βIII-TUB levels were significant in both patient groups compared to controls (**p<0.01, *p<0.05, n=5 experiments for CTR #8 and TSC #6, n = 4 for CTR #5 and TSC #1). Significance was determined via unpaired nonparametric t tests (Mann-Whitney). Plots represent mean values ± SEM.