Extended Data Fig. 10: IPSC-derived co-culture differentiation and NMJ formation.
From: Muscle-derived miR-126 regulates TDP-43 axonal local synthesis and NMJ integrity in ALS models
a) Representative images from the distal (NMJ) compartment IPSCC-derived co-culture of healthy (KOLF) motor neurons and skeletal muscles. Blue indicates NFH, gray indicates BTX, magenta indicates Titin. White dashed line indicates NMJ region shown in inset. Scale bar: 20 µm. b-c) Quantitative analysis of axonal pTDP-43 fraction (b) and axonal degeneration index (c) in iPSC-derived co-cultures of SOD1A5V, TDP-43M337V, and their isogenic control (KOLF). Data in (b) are shown in violin density plots of pTDP-43 puncta area out of NFH area of axons at the NMJ compartment with marking of first, median, and third quartiles. n = 21; 26, 18 imaging fields from 3 independently grown neuromuscular co-cultures. One-way ANOVA with Holm-Sidak correction. **p = 0.0064, ****p = 4.4 × 10−10. Data in (c) are shown as the mean NFH degeneration index of axons in the NMJ compartment ± SD. n = 28; 23; 18 imaging fields from 3 neuromuscular co-cultures. One-way ANOVA with Holm-Sidak correction. ****p = 4.66 × 10−5(SOD1A5V), 1.96 × 10−5(TDP43M337V). d) Representative image for lentivirus-infected IPSC-MN and IPSC-muscles in co-culture within MFC. Green in IPSC-MN (upper compartment) indicates pLV-hSyn-miR126-GFP, Green in IPSC-muscles (lower compartment) indicates pLV-CMV-miR126-GFP. Scale bar: 150 µm. e) Representative immunofluorescent images for pTDP-43 nuclear localization in SOD1A5V and TDP-43M337V IPSC-MN in the proximal MFC compartment of co-cultures infected with either pLV-hSyn-GFP or pLV-hSyn-miR126-GFP. Blue indicates NFH, gray indicates pTDP-43. White dashed square marks the IPSC-MN magnified in the inset. Scale bar: 20 µm. For a,d,e experiments repeated in 3 neuromuscular co-cultures.
