Stem Cell Therapies For Batten Disease

巴顿病的干细胞疗法

• 批准号: 6895156
• 负责人: MARK D KIRK
• 金额: $ 23.74万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6895156
• 关键词: biotechnology; cell biology; confocal scanning microscopy; electrophysiology; electroretinography; embryonic stem cell; esterase; gene expression; gene therapy; genetic manipulation; green fluorescent proteins; immunoelectron microscopy; insulinlike growth factor; laboratory mouse; nervous system disorder therapy; neural degeneration; neuronal ceroid lipofuscinosis; nonhuman therapy evaluation; phenotype; retina degeneration; stem cell transplantation; vision

DESCRIPTION (provided by applicant): The long-term goal of our research is to develop therapeutic applications for stem cell transplantation to treat hereditary neurodegenerative diseases. Our emphasis is on the neuronal ceroid-lipofuscinoses (NCLs). The NCLs are a group of inherited, progressive degenerative disorders of childhood characterized by loss of nerve cells in the retina and central nervous system (CNS). One NCL is commonly known as Batten Disease. The focus of this proposal is on degeneration in the retina, but our results will be applicable to degeneration throughout the CNS. We will transplant neuralized mouse embryonic stem (ES) cells that express enhanced green fluorescent protein (EGFP; for cell tracing) into the eyes of mouse models for NCLs. Our primary hypothesis is that transplantation of specifically engineered, neural-induced ES cells will retard or prevent retinal degeneration resulting in visual recovery. To test our hypothesis, the following specific aims will be performed. Specific Aim #1 is to induce (neuralize) and transplant ES cells into host eyes and assess stem cell survival and integration within the retina. After induction, the ES cells will be injected into the eyes of mutant mice, and the fate of transplanted cells will be evaluated with antibodies specific for neural and glial markers. Specific Aim #2 is to test in vitro for cross-correction of the cellular phenotype, due to factors secreted by donor ES cells. To promote survival of host neurons, ES cells will be engineered to express a growth factor (IGF-1) or the normal enzyme that is mutated in the Clnl mutant mouse. Use of a CMV promoter will ensure over-expression of the genes by neuralized ES cells. Specific Aim #3 is to assess transplanted cell function and whether host retinal cells exhibit enhanced survival after stem cell transplantation. To determine if a normal ultrastructural phenotype is restored in host retinal cells and whether cellular connections are established between transplanted stem cells and host retinal cells immunoelectron microscopy (using an EGFP antibody), and electrophysiology will be performed. We will use electroretinograms to test for maintenance and/or recovery of visual function, intracellular recordings and dye-injections in retinal slice preparations will be used to determine the functional fates of donor cells as well as that of host cells. This research will provide essential new information about the use of stem cells in potential therapies for the NCLs as well as for other inherited neurodegenerative disorders of the CNS.

描述（由申请人提供）：我们研究的长期目标是开发干细胞移植治疗遗传性神经退行性疾病的治疗应用。我们的重点是神经元蜡样质脂褐质（NCL）。 NCL 是一组遗传性、进行性儿童退行性疾病，其特征是视网膜和中枢神经系统 (CNS) 神经细胞的损失。一种 NCL 通常称为巴顿病。该提案的重点是视网膜变性，但我们的结果将适用于整个中枢神经系统的变性。我们将把表达增强型绿色荧光蛋白（EGFP；用于细胞示踪）的神经化小鼠胚胎干 (ES) 细胞移植到 NCL 小鼠模型的眼睛中。我们的主要假设是，移植专门设计的神经诱导 ES 细胞将延缓或防止视网膜变性，从而导致视力恢复。为了检验我们的假设，将实现以下具体目标。具体目标#1 是诱导（神经化）ES 细胞并将其移植到宿主眼睛中，并评估干细胞在视网膜内的存活和整合。诱导后，ES细胞将被注射到突变小鼠的眼睛中，移植细胞的命运将用针对神经和神经胶质标记物的特异性抗体进行评估。具体目标#2 是在体外测试由供体 ES 细胞分泌的因子引起的细胞表型的交叉校正。为了促进宿主神经元的存活，ES细胞将被改造为表达生长因子（IGF-1）或在Clnl突变小鼠中突变的正常酶。使用 CMV 启动子将确保神经化 ES 细胞过度表达基因。具体目标#3是评估移植细胞的功能以及干细胞移植后宿主视网膜细胞是否表现出更高的存活率。为了确定宿主视网膜细胞中是否恢复了正常的超微结构表型以及移植的干细胞和宿主视网膜细胞之间是否建立了细胞连接，将进行免疫电子显微镜（使用 EGFP 抗体）和电生理学检查。我们将使用视网膜电图来测试视觉功能的维持和/或恢复，视网膜切片制剂中的细胞内记录和染料注射将用于确定供体细胞以及宿主细胞的功能命运。这项研究将为 NCL 以及其他中枢神经系统遗传性神经退行性疾病的潜在疗法中使用干细胞提供重要的新信息。