Summary
Two-dimensional (2D) human skeletal muscle fiber cultures are ill equipped to support the contractile properties of maturing muscle fibers. This limits their application to the study of adult human neuromuscular junction (NMJ) development, a process requiring maturation of muscle fibers in the presence of motor neuron endplates. Here we describe a three-dimensional (3D) co-culture method whereby human muscle progenitors mixed with human pluripotent stem cell-derived motor neurons self-organize to form numerous functional NMJ connections within two weeks. Functional connectivity between motor neuron endplates and muscle fibers is confirmed with calcium transient imaging and electrophysiological recordings. Notably, we find epsilon acetylcholine receptor subunit protein expression and activity in our 3D co-culture demonstrating that the system supports a developmental shift from the embryonic to adult form of the receptor not previously reported in 2D culture. Further, co-culture treatment with myasthenia gravis patient sera demonstrates the ease of human disease studies using the system. This work provides the first method to model and evaluate adult NMJ development and disease in culture.