Fig. 7: Axonal mitochondrial behavior and muscle spindle generation in untreated and targeted patient DRGOs.

a Illustration of the microfluidic device with the two lateral channels connected by a set of horizontal microgrooves of length 935 µm, 5 µm width and 6 µm height. The proximal lateral channel includes two open chambers where DRGOs can be contained and cultured. b Representative image of DRGOs seeded in the lateral chambers of the microfluidic device. Scale bar, 1 mm. c Representative images of DRGO axons within the microgrooves stained for TOMM20 (red) and NF200 (green) to visualize the number and morphology of mitochondria. Scale bar, 5 μm. d–f Analysis of number (d), size (e) and circularity (f) of TOMM20 + mitochondria in axons of control, PTL6 and PTL6-LD3 DRGOs. Mean ± s.d., n = 5 independent experiments, 4–6 organoids/line/experiment. *P < 0.05; **P < 0.01; one-way ANOVA with Bonferroni correction. g Representative electron microscopy images showing the morphology of axonal mitochondria (red). Scale bar, 1 μm. h, i Representative immunofluorescence (h) and quantitative analysis (i) of muscle spindles in 4 weeks co-cultures between intrafusal muscle fibers and DRGOs from control (CTRL), PTL6 and isogenic PTL6-LD iPSCs. Mean ± s.d., n = 3 independent experiments, 4 organoids/line/experiment. ***P < 0.001; one-way ANOVA with Bonferroni correction. Scale bar: 100 μm. j, k Immunofluorescence for the synaptic vesicle marker Synapsin (green) within axonal NF200 (red) (j) and quantification of Synapsin puncta (k) on PTL6 and its isogenic line. Mean ± s.d., n = 3 independent experiments 3 organoids/line/experiment. *P < 0.05; **P < 0.01; one-way ANOVA with Bonferroni correction. Scale bar, 5 μm.