Functional cortical axon tracts generated from human stem cell-derived neurons.

Axon tracts support connectivity among different brain regions, which is crucial for the proper functioning of the brain. It is well known that axon regeneration in the central nervous system is poor, and thus, axonal injury often leads to persistent neurological and cognitive deficits. A novel approach to this problem is the transplantation of preformed axon tracts engineered in a laboratory setting. In this study, we describe a potential human substrate for this repair strategy: cortical axon tracts generated from human pluripotent stem cell-derived neurons using the technique of axon stretch growth. Human cortical neurons were differentiated from embryonic stem cell or induced pluripotent stem cell lines. Subjecting these neurons to a stretch growth protocol resulted in the formation of 1-cm-long axon tracts in less than 1 month. Stretch rates of up to 1 mm/day were achieved. These constructs consisted of two clusters of neuronal somata connected by dense intervening axons. The neuronal clusters were composed of primarily infragranular cortical neurons, although some supragranular neurons also were observed. Individual axons exhibited spontaneous calcium waves, illustrating their functionality. Our findings demonstrate the feasibility of engineering functional cortical axons from human stem cell sources, raising the possibility of an autologous approach for cerebral axon transplantation after injury. These constructs also provide a resource for in vitro studies into the dynamics of modular networks and for disease modeling purposes. Impact Statement Axon regeneration is negligible in the adult mammalian brain, and thus, white matter damage often leads to permanent neurological deficits. A novel approach for axon repair is the generation of axon tracts in the laboratory setting followed by transplantation of these constructs. This article details a human substrate for this repair strategy. Using the technique of axon stretch growth, functional cortical axon tracts are generated from human pluripotent stem cells at rates of up to 1 mm/day. These results form the basis of a potential patient-specific protocol for cerebral axon transplantation after injury.