Project Summary/Abstract Multiple sclerosis (MS) is a degenerative disease, which affects the central nervous system (CNS). While most neurodegenerative diseases affect older populations, the onset of MS generally occurs early in life. There is no cure for MS. Currently used drugs have severe side effects. Past attempts to develop cell therapies to treat MS have met with limited success. The major challenges in developing cell therapies include invasive isolation techniques, limited growth and differentiation potential, as well as genetic instability of adult mesenchymal stem cells (MSCs). We have isolated and differentiated highly proliferative and primitive (p) MSCs into neural stem cells (NSCs). In our preliminary studies, transplantation of NSCs significantly reversed the clinical symptoms when transplanted at an early stage of experimental autoimmune encephalomyelitis (EAE) in a mouse model. These findings are very promising and provide a strong “proof of concept” for cell-based treatment of MS. Since we saw substantial improvement in EAE disease with a single cell dose, we hypothesize that multiple doses of NSCs will be more effective in ameliorating chronic EAE disease symptoms and promoting functional recovery. We envision that this innovative approach using NSCs will enhanced the potency of cell therapy as proposed in this study. The specific aims are: 1. To determine the therapeutic effects of repeated doses of NSCs on chronic EAE in mice. We hypothesize that similar to repeated use of drugs, repeated cell therapy treatments will be more efficacious. This hypothesis will be tested by injecting GFP-labeled NSCs in 3 doses to counter chronic EAE induced by MOG immunization in mice. Changes in the disease symptoms and progression will be monitored by performing neurobehavioral, neurological motor function, mechanical threshold response and cold response analyses to assess the effect of cell therapy on the disease progression and remission. 2. To investigate CNS pathology at cellular and molecular levels in NSC transplanted EAE mice. We hypothesized that NSC treatments will reduce inflammation and restore CNS function. Histopathological analysis of the CNS will be performed to access immune cell infiltrates. Composition of cell infiltrates will be assessed by immunohistochemical analysis of CNS sections. Levels of pro- and anti-inflammatory cytokines will be carried to assess the immunomodulatory properties of transplanted NSCs. NSCs are also likely to help in mitigating the imbalance of immune regulatory cells, reduce astrogliosis, and improve myelination. This will be investigated using appropriate cellular and molecular techniques. NSCs also express high level of neurotrophic factors, their role in neuroprotection will be explored. The effect of NSCs on the global gene expression in the CNS of EAE mice will be examined by RNA-seq and validated by qRT-PCR analysis. RNA-seq analysis should help in determining the signaling pathways involve...