(A) The induction steps for the in vitro hNMJ formation from hiPSCs and temporal events in the culture system. SV2, synaptic vesicle protein 2; BDNF/GDNF, brain-derived neurotrophic factor/glial cell–derived neurotrophic factor; NT3, neurotrophin-3. (B) Morphologies of pre- and postsynaptic features at day 21 and day 60 with z axis views. Tuj1-labeled neurons in the presynaptic phase. AChR is labeled by a-BTX-647. MYH, skeletal muscle marker. (C) The morphology of AChR clustering and an NMJ at day 30 and day 60. NF, neurofilaments. See also Supplemental Figure 1, B and C. Scale bars: 10 μm (B and C).