A 3D culture model of innervated human skeletal muscle enables studies of the adult neuromuscular junction

TLDR

Two‑dimensional human skeletal muscle cultures cannot support mature contractile properties, limiting their use for studying adult neuromuscular junction development that requires mature fibers and motor neuron endplates. This work introduces a simple 3D co‑culture method that lets human muscle progenitors and pluripotent stem cell‑derived motor neurons self‑organize into functional neuromuscular junctions, enabling de novo modeling of adult NMJ development or disease. Human muscle progenitors are mixed with pluripotent stem cell‑derived motor neurons in a 3D scaffold, allowing self‑organization into functional NMJ connections. Functional connectivity was confirmed by calcium imaging and electrophysiology, with selective upregulation of epsilon acetylcholine receptor subunits and responsiveness to myasthenia gravis patient sera, demonstrating the system’s utility for disease modeling.

Abstract

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 functional NMJ connections. Functional connectivity between motor neuron endplates and muscle fibers is confirmed with calcium imaging and electrophysiological recordings. Notably, we only observed epsilon acetylcholine receptor subunit protein upregulation and activity in 3D co-cultures. Further, 3D co-culture treatments with myasthenia gravis patient sera shows the ease of studying human disease with the system. Hence, this work offers a simple method to model and evaluate adult human NMJ de novo development or disease in culture.