Fig. 5: Generation of an all human SMA soNMJ model.

a Schematic illustration of the soNMJ differentiation from SMA type I hiPSCs. b Representative images of αBTX clusters in SMA iPSC derived soNMJs and control at day 50 of differentiation. Scale bars: 75 µm. c Quantifications at day 50 of the αBTX⁺ clusters revealed a significant reduction in the number and size of αBTX clusters in the SMA type I soNMJ cultures compared to H1, H9, and XM001 control lines. The statistical tests employed included one-way ANOVA with Bonferroni’s multiple comparison test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. Each dot with the same colour/shape represents the number of NMJs in a well from a single differentiation experiment of a specific cell line. The different colours correspond to different cell lines and the different shapes to different experiments (N). H1: N = 3, n = 9; H9: N = 3, n = 9; XM001: N = 3, n = 9; SMA pt.1: N = 3, n = 9; SMA pt.2: N = 3, n = 9. d Immunofluorescence analysis using Fast MyHC marker at day 50 showed compromised skeletal muscle fiber organization in the two SMA type I hiPSC lines compared to H1 hPSC control. Results are shown as the angle of deviation of each individual fiber from the average fiber orientation. H1: N = 3, n = 9; SMApt1: N = 3, n = 9; SMApt2 N = 3, n = 9. Scale bars: 10 µm. e Skeletal muscle contraction was impaired in SMA type I compared to XM001 control hiPSC line at day 50 of differentiation. H1: N = 3, n = 9; XM001: N = 3, n = 9; SMA pt1: N = 3, n = 9. f Muscle contraction analyzed in SMA type I at day 50 did not respond to 10 μM acetylcholine and 10 μM curare administration compared to H1 hPSC control line. hiPSCs: Human induced pluripotent stem cells; soNMJ: self-organizing neuromuscular junction.