Efficacy of NSCs in an EAE model of MS

项目总结/摘要 多发性硬化（MS）是一种影响中枢神经系统（CNS）的退行性疾病。虽然大多数 虽然神经退行性疾病影响老年人群，但MS的发作通常发生在生命早期。没有 目前使用的药物有严重的副作用。过去开发细胞疗法治疗MS的尝试 取得了有限的成功。开发细胞疗法的主要挑战包括侵入性分离 技术，有限的生长和分化潜力，以及成人间充质干细胞的遗传不稳定性， 细胞（MSC）。我们已经分离并分化了高度增殖和原始的（p）MSCs， 细胞（NSC）。在我们的初步研究中，神经干细胞移植显著逆转了临床症状 当移植到小鼠模型实验性自身免疫性脑脊髓炎（EAE）的早期阶段时。 这些发现非常有希望，并为基于细胞的多发性硬化症治疗提供了强有力的“概念证明”。自 我们看到单细胞剂量对EAE疾病有实质性的改善，我们假设多剂量 神经干细胞在改善慢性EAE疾病症状和促进功能恢复方面将更有效 复苏我们设想，这种使用NSC的创新方法将增强细胞治疗的效力， 提出了一种新的方法。具体目标是：1.为了确定重复剂量的 NSC对小鼠慢性EAE的影响。我们假设，类似于重复使用药物，重复细胞治疗 治疗会更有效。将通过以3个剂量注射GFP标记的NSC来检验该假设， 对抗小鼠MOG免疫诱导的慢性EAE。疾病症状和进展的变化 将通过进行神经行为、神经运动功能、机械阈值反应 和冷反应分析以评估细胞疗法对疾病进展和缓解的作用。2.到 在细胞和分子水平上研究NSC移植EAE小鼠中CNS病理学。我们 假设NSC治疗将减少炎症并恢复CNS功能。组织病理学分析 将进行CNS的检查以获得免疫细胞浸润。将通过以下方法评估细胞浸润的组成： CNS切片的免疫组织化学分析。促炎细胞因子和抗炎细胞因子的水平将被携带到 评估移植的NSC的免疫调节特性。NSC也可能有助于减轻 免疫调节细胞失衡，减少星形胶质细胞增生，并改善髓鞘形成。这将被调查 使用适当的细胞和分子技术。神经干细胞还表达高水平的神经营养因子， 神经保护的作用将被探索。神经干细胞对EAE中枢神经系统基因表达的影响 将通过RNA-seq检查小鼠并通过qRT-PCR分析验证。RNA-seq分析应该有助于 确定参与EAE小鼠受损CNS潜在功能恢复的信号通路。的 这项研究的结果将为EAE提供基本的见解，也有助于开发细胞疗法， 不仅适用于MS，也适用于其他神经退行性疾病。

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 involved in potential functional recovery of damaged CNS in EAE mice. The results of this research will provide fundamental insights into EAE and also help in developing cell therapies not only for MS but also for other neurodegenerative diseases.