Neural progenitor cell/neural stem cell (NPC/NSC) grafts integrate into sites of spinal cord injury (SCI) and show anatomical and electrophysiological evidence of forming neuronal relays across lesions. To determine how signals may be propagated through grafts, we performed ex vivo and in vivo calcium imaging of NPC grafts and host spinal cord neurons in mice. Following optogenetic stimulation of corticospinal tract axon terminals ex vivo, we detected consistent calcium responses throughout grafts. Grafts exhibited spontaneous activity in both independently-firing neurons and emergent neuronal networks, which were also activated by corticospinal stimulation. These patterns of activation resembled those present in intact spinal cord. In turn, optogenetic stimulation of graft axons extending out of lesion sites triggered excitatory post-synaptic responses in caudal host spinal cord neurons. Finally, distinct sensory stimuli elicited responses in the dorsal aspect of grafts in vivo. Thus, NPC/NSC grafts form local networks that are activated by host inputs and can activate host neurons on the distal side of spinal cord lesions.

In order to properly assess the potential benefit of neural stem cell grafts in humans, we sought to understand the rate at which human grafts mature in the injury site. We grafted human NSCs into sites of SCI in immunodeficient rats and assessed anatomical and functional outcomes over a 1.5-year period. Although mature neuron markers appeared 3 months after grafting; neurogenesis, neuronal pruning, and neuronal soma enlargement progressed over the next year. Graft size remained stable as axons emerged in large numbers early on, with half of these projections persisting after 1.5 years. Astrocytes expressed mature markers after 6 months, while oligodendrocytes did not display mature markers until 1 year after grafting. A slow migration of astrocytes from graft sites into host tissue was observed. Importantly, functional improvement did not occur until over 1 year after grafting. Thus, human NSCs mature at their intrinsic rate even when grafted into a rodent environment, and they support functional recovery only at this delayed timepoint, a key finding in human clinical trial planning.