Wake Forest Institute for Regenerative Medicine (WFIRM) Winston-Salem , North Carolina, United States
Abstract: The human neuromuscular junction (NMJ) is a specialized synapse that modulates neurological control over muscle contraction, facilitating precise communication between motor neurons, skeletal muscle cells, and terminal Schwann cells. While the neurophysiological properties of the NMJ have been extensively characterized in animal models, our knowledge of human NMJ neurophysiology remains limited. Compared to animal models, human NMJs are smaller, less complex, and more fragmented, with divergent molecular and cellular characteristics. Furthermore, NMJs are vulnerable entry points for pathogens and toxins that can impair motor function and propagate central nervous system (CNS) infections.
To address this, we optimized a protocol for generating functional human NMJs through directed differentiation of human pluripotent stem cells (hPSCs), producing cultures that recapitulate key features of native human NMJs. These cultures form self-organizing bundles of aligned muscle fibers encircled by innervating motor neurons. Detailed imaging demonstrates the presence of skeletal muscles, satellite cells, glia cells, terminal Schwann cells, interneurons, and spinal cord motor neurons. In contrast to monocultures or co-cultures of only two distinct cell types, these cultures reflect the varied composition of cell types seen in vivo. To validate the utility of this platform for investigating neurotoxin-induced pathologies, we characterized the differential paralytic effects of various Botulinum Neurotoxins on synaptic transmission using electrophysiological methods and in vitro imaging.
This model system offers a comprehensive framework for creating targeted therapies to treat neuromuscular dysfunction and advances our knowledge of human NMJ pathophysiology, in addition to evaluating the mechanisms by which infections and toxins impair NMJ function.
Funding Source: This work was supported by the Defense Threat Reduction Agency—Joint Science and Technology Office (Project MCDC2201-002)
