This project is aimed at testing the feasibility of using human embryonic stem cell (hESC)-derived neural stem/progenitor cells (hNPCs) expressing constitutively active MEF2C (MEF2CA) transcriptional factor as a cell therapy candidate for stroke. A rodent stroke model is being tested for behavioral and histological improvement after transplantation with hNPCs stably expressing MEF2CA (designated here as stable-MEF2CA hNPCs). Currently, we are optimizing this therapeutic candidate for most efficient and tolerated dose, and generating scalable quantities of hESC-derived stable-MEF2CA hNPCs. These cells are transplanted into the brain of the spontaneously hypertensive rat (SHR) model of focal stroke. We will then evaluate behavioral and histological improvements, and perform preliminary safety assessments. Leading up to these experiments, during the past year, we completed a direct comparison of two different types of MEF2CA-programmed candidate stem cell products under two expression systems (stable transformant vs. transient AAV-infection) and found that the stable-MEF2CA hNPC line was superior. Specifically, we performed twelve-week behavioral assessments in the living animals and then sacrificed them to conduct histological evaluations of postmortem brain samples. The results from these experiments indicate significant improvement compared to saline-injected animals. Moreover, there was no significant difference in behavioral improvement or in vivo histological analysis between AAV infection-mediated MEF2CA expression vs. stable-MEF2CA hNPCs. In light of these results, and given that the stable-MEF2CA hNPC line consists of homogenous cells that are more amenable for scale-up for future clinical trials, we concluded with CIRM Program approval that we should move forward with the stable-MEF2CA line for the reminder of the studies. In order to determine the optimal therapeutic dose of cells for transplantation, we are currently conducting dose-response experiments by transplanting three doses of stable-MEF2CA-hNPCs into the striatum of our rat stroke model. At the time of this report we are completing these transplantation experiments and are beginning behavioral assessments in these animals.