The human neuromuscular junction (NMJ) is a specialized synapse responsible for modulating neurological influences on muscle contraction and enabling precise motor neuron-to-muscle cell communication. Compared to rodent NMJs, human neurophysiology remains poorly characterized, highlighting the need for more translatable disease models.

To study the NMJ's development and organization in humans, we employed a previously established system of neuromuscular organoids derived from induced pluripotent stem cells. This 3D co-culture system supports the concurrent development of motor neurons and skeletal muscle, exhibiting key molecular and structural hallmarks of NMJ formation, including characteristic synaptic architectures, acetylcholine receptor clustering, and spontaneous contractile activity.

To validate the physiological relevance of this model, we evaluated its response to known neurotoxins that disrupt neuromuscular transmission: botulinum neurotoxin A (BoNT/A), tetrodotoxin (TTX), and curare. Exposure to these agents resulted in reproducible impairments in synaptic communication and contractility, consistent with their known mechanisms of action. These findings demonstrate the model’s sensitivity to both presynaptic and postsynaptic disruption and support its utility for studying NMJ pathology under controlled conditions.

This system enables detailed investigations into the mechanisms underlying NMJ dysfunction and offers a manipulatable platform for screening therapeutic interventions. It advances our ability to replicate human NMJ biology and disease, including disruptions to neurotransmitter release and synaptic signaling, and provides a promising tool for identifying molecular targets in neuromuscular and motor neuron disorders.