Figure 3.

WFS1 cDNA knockin rescues phenotype in WS neurons at DIV 21

(A) Gene editing strategy with CRISPR-Cas9 technology for insertion at the AAVS1 locus of WFS1 cDNA under a doxycycline inducible promoter in WS5 iPSC line.

(B) Immunoblot detection of wolframin in rescued WS5 (WS5R) NSCs and neurons with or without doxycycline (Dox) treatment and in CT2.

(C) HuC/D and CUX2 immunostaining of WS5R neurons treated or not with Dox. Scale bars, 100 μm.

(D) Proportion of HuC/D⁺- and CUX2⁺-immunoreactive cells in WS5R neurons treated or not with Dox. Data are presented as mean ± SEM (n = 3 biologically independent differentiations; Student’s t test).

(E) TUJ1- and HuC/D-immunoreactive WS5R neurons treated or not with Dox. Scale bars, 100 μm.

(F) Quantification of the mean thickness of isolated and bundled neurites for WS5R neurons treated or not with Dox compared to CT1.

(G) Quantification of the number of isolated and bundled neurites per mm² for three thickness intervals (0–3, 3–6, and >6 μm) in neurons.

(H) Analysis of the number of isolated and bundled neurites per mm² for the >6 μm interval in WS5R neurons treated or not with Dox compared to CT1. For (F), (G), and (H), data are expressed relative to that of CT1 cells and presented as mean ± SEM (n = 3 for CT1 and n = 4 for WS5R and WSR5 + Dox biologically independent differentiations; ^∗∗∗p < 0.001; one-way ANOVA with Dunnett’s post hoc test, data are compared to CT1 cells).