Human spinal interneurons repair the injured rat spinal cord through synaptic integration

Spinal cord injury (SCI) results in irreversible disruption of motor, sensory, and autonomic circuits with no approved reparative treatment to restore lost function. Endogenous V2a spinal interneurons (SpINs) have been implicated as a key component in recovery post-SCI, but the integration of these cells with injured host networks after transplantation has been unexplored. Here, we engineered human V2a-enriched SpINs from an optogenetic channelrhodopsin-2 (ChR2) expressing the human induced pluripotent stem cell (hiPSC) line and evaluated their maturation, connectivity, and function after transplantation into a rat model of cervical SCI. We used in vitro multielectrode array recordings to demonstrate spontaneous and light-evoked activity, as well as functional connectivity between V2a-enriched SpINs and spinal motor neurons. After transplantation, pseudorabies virus tracing from the diaphragm revealed donor neurons synaptically connected with the injured phrenic motor network. Leveraging optogenetics and multiunit electrophysiology, we showed that transplanted human SpINs could both receive (host to transplant) and provide (transplant to host) functional synaptic connections with injured motor networks, reflecting a functional neuronal relay. Single-cell and single-nucleus transcriptomic analyses confirmed transplant maturation into multiple excitatory interneuron subtypes and glial populations and identified genes specific to V2a SpINs synapsing with the host phrenic motor network, including SLITRK5 and PLXNA3. This study demonstrates the feasibility of generating human cervical V2a-enriched SpINs that establish functional host-transplant connectivity and improve motor recovery post-SCI.