Fig. 6

Expression of genes specific to neuromuscular junction and innervation of iPSC-derived skeletal myocytes. A Expression of genes related to different components of the neuromuscular apparatus was analyzed to understand effects of neural and endothelial cells on neuromuscular junction proteins. The presence of motor neurons by itself was sufficient to significantly increase expression of neuromuscular genes like LRP4, RAPSYN, and DOK7. However, addition of endothelial cells to neuromuscular cocultures downregulated LRP4 while promoting expression of muscle-specific kinase (MUSK) gene. Ordinary one-way ANOVA analysis with Tukey’s multiple comparison test was performed with n = 3 replicates per group, and p < 0.05 was considered for statistical significance. B–C Human iPSC-derived motor neurons were observed to innervate skeletal myocytes in the B MYO + MN and C MYO + MN + EC groups. B In the MYO + MN group, points of innervation were identified upon colabelling for presynaptic marker, synaptophysin (red), and acetylcholine receptor-specific bungarotoxin (purple) indicating putative NMJs. C Similarly, in the triculture group (MYO + MN + EC), we found axons running parallel to and innervating myofibers. Points of contact between axon terminal and myofiber is demarcated by white ovals. Rectangular call out boxes denote zoom-in images near points of innervation. Scale bar — 50 μm