Fig. 4

Glia-supported neurite extension method for fluorescently labeled induced pluripotent stem cell (iPSC)-derived neurospheres. a Schema of the glia-supported neurite extension method with fluorescently labeled iPSC-derived neurospheres. b,c Effect of the glia-supported neurite extension method on the fluorescently labeled iPSC-derived neurospheres. After 3 days, the non-polarized cells spread in a disordered manner without a glial cell line; however, using glia-supported neurite extension methods (with GDC90), the differentiating neurites were found to migrate from the neurospheres in a radial and orderly manner (scale bar = 300 μm). Rapid and straightforward extension of the neurites was easily observed using strong EGFP fluorescence. d Fluorescence and phase contrast merged images of the glia-supported neurite extension method using EGFP-labeled iPSC-derived neurospheres and tdTomato-labeled human glial GDC90 cells (scale bar = 200 μm). e Immunostaining for the young neuron marker doublecortin (DCX) showed that the neurites extending on the tdTomato-positive human glial cells were also stained with EGFP (scale bar = 50 μm). f–k EGFP fluorescence of the extending neurites observed using the glia-supported neurite extension method on day 5 after plating on the dish. Patient A (p.N329S)-derived neural progenitor cells (NPCs) showed poor neurite extension (f–g) compared to the Patient B (p.R264C)-derived (h-i) or healthy control iPSC-derived NPCs (j–k) (scale bar = 200 μm). l Length of EGFP-positive neurites extending from the neurospheres (μm: mean ± SEM). Boxplot graph illustrating median, first quartile, and third quartile with each outliner plotted. (**p < 0.01, one-way ANOVA followed by Dunnett’s test. n = 269, 277, 374, 369, 338, and 213 in A#1, A#3, B#1, B#2, 201B7 (control), and UCCiPS1#1, respectively. Statistical analyses were performed using 201B7 as the control. When using ONH-UCCiPS1 independently as a control, the same results were also confirmed.). m, n Magnified images of coronal scanned MR images showing the morphological differences in the corpus callosum between Patient A (T2-weighted) and Patient B (T1-weighted). The corpus callosum was not present in the MR images (M) of Patient A (with the p.N329S mutation), suggesting that the interhemispheric projection was hypoplastic, unlike that in Patient B (with the p.R264C mutation). Poor neurite extension of patient-derived iPSCs was considered to correspond to the patient’s phenotype and severity of the disease