Engineering Spinal Interneurons for Repair of the Injured Cervical Spinal Cord

Cervical spinal cord injuries (SCI), the most common SCI, compromise respiratory networks leading to life‐threatening impairments in breathing. Following a cervical injury, extensive damage occurs to the phrenic motor network (controlling the diaphragm) consisting of phrenic motoneurons and interneurons. Despite these devastating consequences, there is now significant evidence for plasticity and limited spontaneous recovery. Our ongoing work has identified spinal interneurons (SpINs), in particular the excitatory V2a subtype, as key therapeutic targets for promoting plasticity and functional recovery of the phrenic motor network. Efforts to enhance this plasticity have shown some promise, but it is likely that treatments capable of promoting repair (e.g., cell transplantation) will provide the best therapeutic outcome. Building on a long history of transplanting spinal neural precursor cells (NPCs) to promote spinal cord repair, the present work harnesses advances in stem cell engineering to enrich donor NPCs with specific subsets of stem cell derived SpINs. We hypothesize that NPCs enriched for V2a SpINs will have enhanced therapeutic efficacy over NPCs alone and promote phrenic motor recovery. We will test this hypothesis using a clinically relevant model of a cervical contusion in the adult rat, and test the functional contribution of donor V2a SpINs using inhibitory chemogenetics (hM4Di). Adult female Sprague‐Dawley rats received mid‐cervical contusion injury (Infinite Horizon impactor, intended impact force 200 kilodyne). One‐week post‐injury, aggregates of NPCs with mouse embryonic stem cell derived V2a SpINs were injected into the lesion cavity. One month post‐transplantation, animals were re‐anesthetised and traced with a retrograde, transneuronal tracer, pseudorabies virus, applied to the diaphragm muscle to assess donor‐host connectivity with the phrenic network. Three days later, animals underwent terminal diaphragm electrophysiology, at which time clozapine was injected intraspinally into the transplant to silence donor SpINs. Preliminary data revealed that clozapine‐induced suppression of donor cell activity reduced diaphragm activity ipsilateral to injury, with no measurable effect on the contralateral side. Consistent with this, immunohistochemical assessment revealed donor cells survived and synaptically integrated with the injured host phrenic network. This ongoing research offers the first in‐depth assessment of how these donor SpIN populations contribute to phrenic recovery.