Derived-motor neuron transplantation is associated with nerve regeneration in a chronically denervated nerve with delayed adoption of a chronic denervation phenotype

JOURNAL/rmrep/04.03/02273995-990000000-00037/figure1/v/2026-05-22T144156Z/r/image-tiff Objectives: After acute injury, the peripheral nervous system regenerates more efficiently than the central nervous system; however, this advantage is not maintained with chronic injury. In large animals such as humans, the slow rate of axon growth and the long distance between the central nervous system and end organ result in the adoption of a chronically denervated nerve phenotype even in the setting of acute injury: distal areas of the nerve become unable to support regenerating axons, even as the proximal aspect of acute injury may be appropriately supportive of regeneration. We hypothesized that motor neuron transplantation into the denervated distal stump would maintain regenerative capacity by effectively reducing the time and distance necessary for regeneration through relay formation and by supporting the host Schwann cells responsible for guiding regenerating axons. Methods: Motor neurons were derived from mouse embryonic stem cells. These cells were tested for synaptogenic potential through a co-culture model using synaptic staining and calcium imaging, with attenuation by various inhibitors of synaptic transmission. Additionally, these cells were transplanted (along with dead cells and vehicle controls) into a 1-week denervated nerve and allowed to engraft for 2 or 3 months prior to nerve repair. Electrodiagnostic, immunofluorescence staining, gene expression, nerve ultrastructure, and muscle morphometry were used to measure the recovery of the motor response and to evaluate in vivo synapse formation and host Schwann cell health. Results: Using in vitro assays, we demonstrated the feasibility of relay formation by demonstrating ventral horn to derived-motor neuron synaptogenesis as identified by calcium imaging. Pilot studies of the transplantation of embryonic stem cell-derived mouse motor neurons into chronically denervated rat nerves demonstrated improved regenerative capacity in the chronically denervated nerve following a delayed repair paradigm. While host-to-graft synaptogenesis was not observed, the Schwann cells in the transplanted nerve stump could activate a pro-regenerative state despite chronic denervation. Tumorigenesis occurred in one-third of live cell transplants and limited the statistical power of the study by reducing the sample size. Conclusion: These data suggest that cell transplantation is associated with a delay in the adoption of a chronically denervated phenotype, primarily by maintaining the ability of Schwann cells to maintain a pro-regenerative state.