Efficacy of NSCs in an EAE model of MS

项目摘要/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。虽然大多数人 神经退行性疾病影响老年人群，MS的发病一般发生在生命的早期。没有 治愈多发性硬化症目前使用的药物有严重的副作用。过去尝试开发治疗多发性硬化症的细胞疗法 都取得了有限的成功。发展细胞疗法的主要挑战包括侵入性分离。 成体间充质干细胞的技术、有限的生长和分化潜力以及遗传不稳定性 细胞(MSCs)。我们分离并分化出高增殖和原始的(P)间充质干细胞为神经干细胞。 细胞(NSCs)。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。 在实验性自身免疫性脑脊髓炎(EAE)小鼠模型的早期移植。 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“概念证据”。 我们看到EAE疾病在单细胞剂量下有实质性的改善，我们假设多个剂量 神经干细胞将更有效地改善慢性EAE症状，促进功能 恢复。我们设想，这种使用神经干细胞的创新方法将增强细胞治疗的效力，因为 在本研究中提出。其具体目的是：1.确定重复剂量的治疗效果 神经干细胞对小鼠慢性EAE的影响。我们假设，类似于重复使用药物，重复细胞疗法 治疗将会更加有效。这一假设将通过注射3剂GFP标记的神经干细胞来检验 抗MOG免疫所致小鼠慢性EAE。疾病症状和进展的变化 将通过执行神经行为、神经运动功能、机械阈值反应进行监测 以及冷反应分析，以评估细胞治疗对疾病进展和缓解的影响。2.至 从细胞和分子水平研究神经干细胞移植后EAE小鼠中枢神经系统的病理变化。我们 假设NSC治疗将减少炎症并恢复中枢神经系统功能。组织病理学分析 中枢神经系统将被执行，以获得免疫细胞的渗透。细胞渗出物的成分将通过以下方法进行评估 中枢神经系统切片的免疫组织化学分析。促炎和抗炎细胞因子的水平将被携带到 评估移植的神经干细胞的免疫调节特性。神经干细胞也可能有助于缓解 免疫调节细胞失衡，减少星形胶质细胞增多，改善髓鞘形成。这件事将被调查 使用适当的细胞和分子技术。神经干细胞也表达高水平的神经营养因子，它们的 在神经保护中的作用将被探索。神经干细胞对EAE中枢神经系统基因表达的影响 小鼠将接受RNA-seq检测，并通过qRT-PCR分析进行验证。RNA-seq分析应该有助于 确定参与EAE小鼠受损中枢神经系统潜在功能恢复的信号通路。这个 这项研究的结果将为EAE提供基本的见解，并有助于开发细胞疗法 不仅适用于多发性硬化症，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.