Fig. 3: Formation of functional NMJs in the soNMJ model.

a Schematic illustration of the strategy used to generate the soNMJ model from hPSCs under adherent culture conditions. b High-content imaging of whole well at day 50 soNMJ model. The representative image depicts the self-organization of the neurons TUBB3⁺ (cyan) and skeletal muscle fibers that express Fast MyHC (magenta) in 20 fields acquired in the same well. Scale bars: 1 mm and 100 µm. H1: N = 1; H9: N = 1; XM001: N = 1. c Representative immunofluorescence image at day 50 and day 100 of differentiation showing the presence of TUBB3⁺ neurites in contact with α-bungarotoxin⁺ (αBTX⁺) AChR clusters on fast-twitch skeletal muscle fibers (Fast MyHC⁺) revealing the presence of neuromuscular junctions. Scale bars: 25 µm. d Quantification of the number of αBTX clusters at day 50 and day 100 of hPSC differentiation normalized to the number of Fast MyHC myofibers. The number of NMJs increased significantly from day 50 (H1: 0,419 ± 0,048; H9: 0,435 ± 0,067; XM001: 0,447 ± 0,051) to day 100 (H1: 0,832 ± 0,09; H9: 0,872 ± 0,099; XM001: 0,861 ± 0,059). The statistical tests employed included unpaired t-test with Welch’s correction. *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. Source data are provided as a Source Data file. e At day 100 of differentiation, there was a spontaneous contraction of the skeletal muscles. Contraction analysis by live imaging showed that muscle contraction was increased by administering 10 μM acetylcholine and blocked by adding the acetylcholine receptor inhibitor, curare (10 μM). H1: N = 3, n = 9; XM001: N = 9, n = 27. soNMJ: self-organizing neuromuscular junction. Source data are provided as a Source Data file.