Primary myoblasts were isolated from skeletal muscles of P0.5 mouse pups and differentiated into myotubes as previously described (10 (link)). Myotubes were resuspended in neuronal culture medium and then plated onto coverslips with co-cultured astrocytes and hiMNs at 40 to 50 dpi. After another 4 to 7 days, these sandwich cultures of myotubes, hiMNs, and astrocytes were live-stained with rhodamine-conjugated α-BTX (Invitrogen, 1:10,000) for 1 hour at 37 . α-BTX labelled cells were then processed for immunostaining with antibodies of SYN1 (Cell Signaling Technology, 1:500) and MHC (Sigma, 1:1,000). NMJ formation frequency was presented as the percentage of NMJs on myotubes associated with hiMNs networks.