Fig. 3.

Differentiation of iPSCs from SMA patients into motor neurons. (A to F) After differentiation of iPSCs (HET-iPSCs, corrected and untreated SMA-iPSCs, TR, and SMA, respectively), the following motor neuron markers were expressed: SMI32- (green) and HB9-positive (red) (A to C) or ChAT (red) (yellow represents the merging of red and green colors). (D to F) Double-positive motor neurons were observed (merged, yellow). Nuclei are labeled with DAPI (blue). (G to L) Morphometric analysis of motor neurons. (G and H) Quantification of motor neurons at 5 (G) and 8 weeks (H) after differentiation from iPSCs, showing a reduced number of untreated SMA-iPSC–derived motor neurons compared to corrected SMA-iPSC–derived motor neurons (one-way ANOVA followed by a Tukey’s post hoc test) (*P < 0.001, 8 weeks). Values represent means ± SEM from five independent experiments performed in triplicate. (I to L) At 8 weeks, untreated SMA-iPSC motor neurons showed smaller cell diameters (J) and shorter axon lengths (L) than did motor neurons from the other three groups TR, WT, and HET (all, P < 0.001; size: SMA versus TR, P = 0.002). The difference was more evident at 8 weeks than at 5 weeks (I to K). Insets represent the respective medians. Five independent experiments performed in triplicate. (M to O) Neuromuscular junctions formed when motor neurons were cocultured with myotubes. The acetylcholine receptors were labeled with α-bungarotoxin (red) and axons with SMI32 (green). These are representative images. (P and Q) We evaluated the number (P) and size (Q) of neuromuscular junctions and found a reduced size and number of neuromuscular junctions in untreated SMA-iPSC motor neurons (SMA; patients 1 and 2) compared to corrected SMA-iPSC or HET-iPSC motor neurons (*P < 0.001, one-way ANOVA followed by a Tukey’s post hoc test). (P) Mean ± SEM, n = 5 independent experiments in triplicate. (Q) Inset represents the respective medians. (R to T) Gems are present in HET-MN (R) and TR-MN (S) but were absent in SMA-MN (T). Scale bars, 100 μm (A to C); 80 μm (D to F); 20 μm (M to O); 50 μm (R to T).