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bioRxiv · 10.1101/2025.10.17.682712

A human iPSC-derived motor neuron-myogenic cell coculture platform to evaluate neuromuscular junction innervation after axon injury and in Spinal Muscular Atrophy

Woolf, C. J.·Rubin, L. L.·Heo, K.·Zeng, X.·Zhang, K.·Chen, K.·Naveen, A.·Powell, R.·Giadone, R. M.·Zhen, S.·Pandey, R.
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Abstract

Traumatic nerve injury is challenging as motor neurons with damaged axons repair slowly, which can lead to muscle degeneration, while in Spinal Muscular Atrophy (SMA), muscle innervation is reduced. While pro-regenerative or neuroprotective compounds have been identified, their specific ability to enhance or restore neuromuscular junction (NMJ) function in patients remains unclear due to a lack of in vitro human models that track axon growth, NMJ innervation and muscle function. We developed a human iPSC-derived motor neuron-myogenic coculture platform that enables real-time monitoring of axon growth NMJ innervation and axon regeneration and muscle activity following axonal injury, and for SMA-derived motor neurons. We identified spontaneous synchronized GCaMP6f muscle activity as a useful functional marker of NMJ formation. Using this platform, we show that blebbistatin, a pro-regenerative non-muscle myosin II (NMII) inhibitor differentially regulates growth cone dynamics in injured versus uninjured motor neurons, resulting in enhanced NMJ reinnervation. This highlights the therapeutic potential of developing pro-regenerative compounds to promote NMJ innervation. We also confirm that NMJ function is reduced in SMA type 0 (prenatal onset) and type I (pediatric onset) patient-derived motor neurons in the coculture. This human stem cell-based framework can be used, therefore, to evaluate pro-regenerative compounds for axon injury and neuroprotective one for neurodevelopmental and neurodegenerative disorders.

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Woolf, C. J., Rubin, L. L., Heo, K., Zeng, X., Zhang, K., Chen, K., Naveen, A., Powell, R., Giadone, R. M., Zhen, S., Pandey, R.. 2025-10-17. A human iPSC-derived motor neuron-myogenic cell coculture platform to evaluate neuromuscular junction innervation after axon injury and in Spinal Muscular Atrophy. https://doi.org/10.1101/2025.10.17.682712

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