Spinal cord injury (SCI) often damages, not only white matter axon tracts that transmits signals to and from the brain, but also the central gray matter, causing segmental loss of interneurons and motor neurons. Transplantation of neural stem cells (NSCs) has the potential to replace lost neurons and glia. These transplanted neurons can form functional relays between spinal segments disconnected by the injury. We have found that human cells mature at a much slower time course than originally anticipated (months and years) compared to rodent cells (weeks to months). The proposed studies will determine whether neuronal differentiation of human NSCs can be accelerated by in vivo by activation of G-protein signaling cascades. Human NSCs will be modified prior to grafting to express Designer Receptor Exclusively Activated by Designer Drugs (DREADDs). DREADDs are mutant G-proteins that are solely activated by the otherwise inert ligand, clozapine N-oxide (CNO). Activation of G?s-DREADD leads to increase in the cyclic AMP-dependent signaling cascade resulting in pro-neuronal gene expression, such as BDNF. Activation of G?q/11-DREADD leads to IP3/DAG and calcium signaling resulting in pro-neuronal gene expression and electrophysiological activity. This proposal will assess the following questions: 1) Which DREADD is more efficient at driving neuronal differentiation in vivo? 2) Does accelerated neuronal differentiation of human NSCs enhance host/graft-connectivity and support behavioral recovery? This work is highly innovative and clinically oriented: 1) We will utilize a contusion type injury that is most applicable to human SCI. 2) We will transplant cells at a clinically relevant sub-chronic timepoint (4-weeks post-contusion). 3) We will employ neural stem cell transplants to replace lost neural cells at the lesion site. 4) We will combine neural stem cell grafting with innovative gene therapy techniques to accelerate neuronal differentiation and maturation of grafted cells.