Human Stem Cell Model of Niemann Pick Type C

Niemann Pick C型人类干细胞模型

• 批准号: 7828398
• 负责人: Lawrence S. Goldstein
• 金额: $ 100万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-15 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7828398
• 关键词: Address; Affect; Alzheimer' s Disease; Animal Model; Apolipoprotein E; Area; Astrocytes; Axon; Basic Science; Biochemical; Cell Death; Cell model; Cells; Cessation of life; Childhood; Cholesterol; Complement component C1s; Complex; Country; Data; Dementia; Development; Diploidy; Disease; Disease model; Economics; Employment; Ensure; Environment; Exhibits; Failure; Fibroblasts; Foundations; Future; Gene Silencing; Genetic Engineering; Genetic Heterogeneity; Genetic Variation; Housekeeping; Human; Human Engineering; Human Pathology; Image; Institution; Intervention; Investments; Kinetics; Knowledge; Label; Lead; Life; Light; Link; Lipids; Lipoidosis; Literature; Lysosomes; Measures; Mediating; Methods; Modeling; Mus; Mutation; Natural regeneration; Nerve Degeneration; Neurodegenerative Disorders; Neuroglia; Neuronal Dysfunction; Neurons; Nuclear Pore Complex; Occupations; Pathologic; Pathway interactions; Patients; Pattern; Phenotype; Physicians; Physiological; Point Mutation; Population; Preparation; Process; Property; Proteins; Protocols documentation; Research; Role; Science; Scientist; Security; Services; Somatic Cell; Stem cells; Sterols; Students; Testing; Therapeutic Agents; Viral; Work; amyloid precursor protein processing; base; cell type; cholesterol analog; cholesterol trafficking; disease phenotype; disease-causing mutation; human disease; human embryonic stem cell; human stem cells; induced pluripotent stem cell; innovation; insight; interest; mutant; nervous system disorder; neuronal cell body; neuronal growth; neuronal survival; new technology; next generation; pluripotency; prevent; public health relevance; research and development; small hairpin RNA; stress-activated protein kinase 1; tau phosphorylation; therapy development; trafficking; uptake

DESCRIPTION (provided by applicant): Niemann Pick Type C (NPC1) is a rare but lethal pediatric dementia caused by a mutation in NPC1, a housekeeping protein residing in the late endosomal compartment with a putative role in cholesterol transport. The result is a severe lipidosis characterized by massive accumulation of lysosomal sterols and other lipids that ultimately cause cell death. The disease is of enormous basic science interest as well as a hallmark model of dysfunctional intracellular cholesterol trafficking and because of its similarities with Alzheimer's disease (AD), which suggest shared underlying mechanisms. To date strategies to model NPC have not yet determined how some mutations of NPC1 can cause neuronal failure in humans for two reasons: i) use of animal models that do not replicate all aspects of human pathology, and ii) a focus on accumulation of cholesterol as the cause of neuronal dysfunction in NPC, which may not be the predominant phenotype in neuronal cell populations. We propose to generate the first human neuronal model of NPC1 by genetic engineering of human embryonic stem cells (hESCs) and reprogramming of somatic cells into human induced pluripotency stem cells (hIPSCs). From a broad perspective our approach will have a significant impact in the stem cell field as it cross validates studies in hIPSCs for the study of neurodegenerative diseases. By conducting parallel analysis of hESC and hIPSC lines we will also address the genetic heterogeneity of NPC, and confirm that pathologic phenotypes found in these cells are specifically due to lack of normal NPC1 function. We will use shRNA mediated silencing of NPC1 and insertional methods of viral reprogramming to generate independent sets of NPC1 knockdown hESC lines, and hIPSC lines reprogrammed from NPC fibroblasts respectively. We will follow strict criteria of characterization of these newly generated lines to ensure they maintain stem cell properties, are genetically stable and replicate basic NPC phenotypes described in the mouse literature. We will use protocols we have developed in my lab to generate populations of pure human neurons that we will study in bulk, pure, and compartmented cultures. We will analyze human neurons derived from NPC hESCs and hIPSCs to test specific predictions that have never been probed in a human neuronal model of the disease; i) we will confirm and expand phenotypes typical of NPC that have not been tested in live human neurons, ii) we will evaluate the viability and differentiation capacity of wild type and NPC1 neurons, iii) we will measure the kinetics of cholesterol and lysosomal trafficking and the role of cholesterol in neuronal growth and survival, and iv) we will explore whether NPC is a cell autonomous disease or if neuronal failure in NPC can be affected by the glial environment. Insights generated from our observations have the potential to drastically increase our understanding of how NPC1 causes neuronal failure in humans and guide efforts that may lead to the development of a cure. UCSD is one of the country's leading research institutions. According to data from the U.S. National Science Foundation, UCSD expended nearly $800 million for research and development during the 2007 fiscal year. This project will add to our institution as an economic and academic engine at the state and national level. Our project will contribute to the continued development of the regional and national economy through research, innovation and job creation. Future economic security will be promoted through the continued employment of a senior physician scientist, a senior research technician, as well as the employment of graduate and undergraduate students. The long term impact of this investment will be the creation of knowledge, the preparation of the next generation of academic workforce, the development of new technologies, opportunities for future research and the use of commercial services in the academic sector that have the potential to yield more jobs. PUBLIC HEALTH RELEVANCE: We propose to create the first human neuronal model of Niemann Pick type C1 from human embryonic stem cells and human induced pluripotent stem cells. We will use control and NPC1 human neurons to study the kinetics of cholesterol trafficking in NPC1 and the role of cholesterol in neuronal survival and regeneration. This work will shed important light on an important childhood neurodegenerative disease and on Alzheimer's Disease as well.

描述（由申请人提供）：Niemann Pick Type C （NPC1）是一种罕见但致命的儿童痴呆症，由NPC1突变引起，NPC1是一种居住在内体晚期腔室的管家蛋白，据推测在胆固醇运输中起作用。结果是严重的脂质病，其特征是溶酶体固醇和其他脂质大量积聚，最终导致细胞死亡。这种疾病具有巨大的基础科学兴趣，也是细胞内胆固醇运输功能失调的标志性模型，因为它与阿尔茨海默病（AD）相似，这表明了共同的潜在机制。迄今为止，模拟鼻咽癌的策略尚未确定NPC1的一些突变如何导致人类神经元衰竭，原因有两个：1)使用的动物模型不能复制人类病理的所有方面，2)关注胆固醇积累作为鼻咽癌神经元功能障碍的原因，这可能不是神经细胞群体中的主要表型。我们提出通过对人胚胎干细胞（hESCs）进行基因工程改造，并将体细胞重编程为人诱导多能干细胞（hIPSCs），生成第一个人类NPC1神经元模型。从广泛的角度来看，我们的方法将对干细胞领域产生重大影响，因为它交叉验证了hipsc研究用于神经退行性疾病的研究。通过对hESC和hIPSC细胞系进行平行分析，我们还将研究NPC的遗传异质性，并证实在这些细胞中发现的病理表型是由于缺乏正常的NPC1功能而引起的。我们将使用shRNA介导的NPC1沉默和病毒重编程插入方法，分别从鼻咽癌成纤维细胞中产生独立的NPC1敲除hESC系和重编程的hIPSC系。我们将严格遵循这些新产生的细胞系的鉴定标准，以确保它们保持干细胞特性，遗传稳定，并复制小鼠文献中描述的基本NPC表型。我们将使用我们在实验室开发的方案来产生纯人类神经元群体，我们将在批量，纯和区隔培养中进行研究。我们将分析来自NPC hESCs和hIPSCs的人类神经元，以测试从未在该疾病的人类神经元模型中探索过的特定预测；我)我们将确认和扩大表型人大没有测试的典型生活人类神经元,2)我们将评估野生型的可行性和分化能力和NPC1神经元,iii)我们将衡量胆固醇和溶酶体的动力学贩运和胆固醇在神经元生长和存活的角色,和(四)我们将探讨人大是一种细胞自动疾病或者是否在人大可以影响胶质神经元失败的环境。从我们的观察中产生的见解有可能大大增加我们对NPC1如何导致人类神经元衰竭的理解，并指导可能导致治疗方法开发的努力。加州大学圣地亚哥分校是美国领先的研究机构之一。根据美国国家科学基金会的数据，加州大学圣地亚哥分校在2007财政年度花费了近8亿美元用于研究和开发。这个项目将使我们的机构成为州和国家层面的经济和学术引擎。我们的项目将通过研究、创新和创造就业机会，为地区和国家经济的持续发展做出贡献。未来的经济保障将通过继续雇用高级医师、科学家、高级研究技术人员以及研究生和本科生来促进。这项投资的长期影响将是知识的创造、下一代学术劳动力的准备、新技术的开发、未来研究的机会以及在学术部门使用有可能产生更多就业机会的商业服务。