Multiple sclerosis (MS) is a complex, debilitating neural disorder associated with significant morbidity and mortality. Disease etiology is a result o unknown environmental and genetic factors, while disease pathology presents as inflammatory injury to the central nervous system (CNS) causing physical incapacity. This damage is a result of autoimmune-mediated destruction of oligodendrocytes causing demyelination. Prolonged demyelination leads to axonal damage occurring either in focal lesion sites and/or widespread throughout the CNS, depending on presentation of the disease. MS is treated with disease-modifying agents that target the immune system in an attempt to reduce the frequency of relapses and delay disease progression. These drugs have had limited success in improving patient outcomes and lack the unmet need to promote immediate remyelination. Identifying drugs that induce resident oligodendrocyte progenitor cell (OPC) mediated remyelination would provide patients a vital mechanism to halt disease progression and improve function. We propose to screen drugs that promote remyelination for repurposing in MS. To achieve this goal we have developed a proposal that incorporates the skill and expertise of multiple disciplines through a proven validation pipeline to interrogate drug entities that promote remyelination. We will begin with drugs that have a history of use in human clinical trials and assess their ability o promote rodent OPC maturation and myelin gene expression in our novel high throughput in vitro system. This system takes advantage of our technology to generate scalable sources of functional OPCs from pluripotent stem cells. Validated drug hits from our high throughput in vitro screen will then be tested in a tractable and economical ex vivo myelination assay utilizing organotypic rodent brain slice cultures. Moving forward, we will take our 5 most promising drug hits (as determined from in vitro and ex vivo assays) and test them in animal model systems regularly used to interrogate applicability to MS (lysolecithin-mediated spinal cord demyelination and Experimental Autoimmune Encephalomyelitis). Top drug hits will also be tested on human OPCs. Using our strengths in preclinical MS research and stem cell differentiation, we aim to identify efficacious pharmacological agents that promote remyelination by the end of this two year project. We are prepared to progress lead drugs identified from our studies to human clinical trials.