This new application represents a continuation of the California Primate Spinal Cord Research Consortium project on human embryonic stem cell (hESC) derived neural stem cell transplantation; our overarching goal is to understand both basic mechanisms of spinal cord organization and function in non-human primates, and to leverage these and other advances in developing translational human pro-regenerative therapies. We have made considerable progress in the last 5 years and now propose new specific aims that will directly bear on the potential of human neural stem cells (NSCs) to benefit human SCI. Aim 1: Examine Mechanisms Underlying Graft-Related Functional Improvement: Graft Silencing with Inhibitory DREADDs. Work in this aim will establish whether functional recovery observed in non-human primates after grafts of spinalized neural stem cells is related to graft activation of host neural circuits. This knowledge will be important for human translation. Aim 2: Determine Whether 4-AP Improves Anatomical and Functional Outcomes After Neural Stem Cell Grafts There is a vast literature supporting potentially beneficial effects of 4AP in SCI, and 4AP is approved for human use as a conduction-enhancing drug in multiple sclerosis. This aim will determine whether treatment with neural stem cell grafts plus 4AP improves anatomical and functional outcomes. Aim 3: Determine Whether PTEN/SOCS3 Inhibition Improves Host Corticospinal Regeneration and Functional Outcomes. Rodent studies indicate that functional benefits of neural stem cell grafts are mediated by formation of host-to-graft-to-host electrophysiological relays across sites of SCI. These relays require host axon regeneration into grafts. In humans, corticospinal axons are essential for voluntary movement. This aim will determine whether inactivation of PTEN/SOCS3 will enhance corticospinal axon regeneration and improve functional outcomes in primates.