Fig. 5: Modeling neuromuscular pathology in motor assembloids by IH exposure.

a Representative immunofluorescence images for axons (TUJ1; left), AChR clusters (α-BTX; middle), and myofibers (SAA; right) within motor assembloids. b Displacement traces depicting contraction amplitudes in motor assembloids under electrical stimulation over 4 days of IH. c Schematic detailing the single displacement trace of muscle motion capturing contraction, peak, and relaxation phases in response to electrical stimulation. d Quantification of normalized peak amplitude during 4 consecutive days of IH compared to Ctrl. n = 4 assembloids. e Quantification of a single displacement trace of muscle motion over 4 days of IH, the orange dashed line denotes the peak amplitude of Ctrl. n = 4 assembloids. f and g Representative immunoblots and heatmap profiles showing muscle contractile proteins (f) and neurofilament proteins (g) over 4 days of IH, with β-tubulin and Ponceau S staining as loading controls, respectively. n = 3 independent experiments. h Schematic of MEA recording, from baseline, through optogenetic stimulation (opti-stim), to post-optogenetic stimulation (post-stim). i Plate map of spontaneous mean firing rate (Hz) in hMNS within motor assembloids across 4 days of IH. n = 4 assembloids. j Quantification of spontaneous spike activity in hMNS within motor assembloids across 4 days of IH. n = 4 assembloids. k and l Representative electrical activity mapping of a pair of motor assembloids illustrating variations in spike events (k) and mean spike amplitude (l) per min from Ctrl to IH 4 d. m and n Quantification of electrophysiological parameters, including the mean spike count (m) and mean spike amplitude (n) per min, recorded from electrodes in hMNS and hSkM regions within motor assembloids across Ctrl and IH 4 d. n = 4 assembloids. o Schematic overview of the dynamic changes in phenotypic and functional characteristics of motor assembloids in response to IH. Data are presented as mean ± SD (d, f, g, j) or ± SEM (m, n). Statistical analysis: d Repeated ANOVA with Tukey multiple comparison. f, g, j One-way ANOVA with Bonferroni multiple comparison. m, n two-tailed Student’s t test. ***p  <  0.001, **p <  0.01, *p  <  0.05. Source data are provided as a Source Data file.