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（由申请方提供）：尼曼匹克C型（NPC 1）是一种罕见但致命的儿科痴呆症，由NPC 1突变引起，NPC 1是一种存在于晚期内体区室中的管家蛋白，在胆固醇转运中起假定作用。结果是严重的胆固醇沉积症，其特征是溶酶体固醇和其他脂质的大量积累，最终导致细胞死亡。该疾病具有巨大的基础科学兴趣，也是功能失调的细胞内胆固醇运输的标志性模型，并且由于其与阿尔茨海默病（AD）的相似性，这表明了共同的潜在机制。迄今为止，NPC建模策略尚未确定NPC 1的一些突变如何导致人类神经元衰竭，原因有两个：i）使用不复制人类病理学所有方面的动物模型，以及ii）关注胆固醇的积累作为NPC中神经元功能障碍的原因，其可能不是神经元细胞群中的主要表型。我们建议通过人胚胎干细胞（hESC）的基因工程和将体细胞重编程为人诱导多能性干细胞（hIPSC）来产生NPC 1的第一个人神经元模型。从广泛的角度来看，我们的方法将在干细胞领域产生重大影响，因为它交叉验证了hIPSC用于神经退行性疾病研究的研究。通过对hESC和hIPSC细胞系进行平行分析，我们还将解决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NPC 1功能所致。我们将使用shRNA介导的NPC 1沉默和病毒重编程的插入方法来分别产生独立的NPC 1敲低hESC系和从NPC成纤维细胞重编程的hIPSC系。我们将遵循这些新生成的细胞系的严格表征标准，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协议来生成纯人类神经元群体，我们将在批量，纯和分区培养中进行研究。我们将分析来自NPC hESC和hIPSC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测的特定预测; i）我们将确认和扩增尚未在活的人神经元中测试的NPC典型表型，ii）我们将评估野生型和NPC 1神经元的活力和分化能力，iii）我们将测量胆固醇和溶酶体运输的动力学以及胆固醇在神经元生长和存活中的作用，以及iv）我们将探索NPC是否是细胞自主性疾病或者NPC中的神经元衰竭是否可以受到神经胶质环境的影响。从我们的观察中产生的见解有可能大大增加我们对NPC 1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发展的努力。UCSD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，UCSD在2007财政年度花费了近8亿美元用于研发。该项目将增加我们的机构作为一个经济和学术引擎在国家和国家一级。我们的项目将通过研究，创新和创造就业机会，为区域和国家经济的持续发展做出贡献。未来的经济安全将通过继续雇用一名高级医生科学家、一名高级研究技术员以及雇用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，未来研究的机会和学术部门商业服务的使用，有可能产生更多的就业机会。 公共卫生关系：我们建议从人胚胎干细胞和人诱导多能干细胞中建立第一个尼曼匹克C1型人类神经元模型。我们将使用控制和NPC 1人类神经元来研究NPC 1中胆固醇运输的动力学以及胆固醇在神经元存活和再生中的作用。这项工作将揭示一个重要的儿童神经退行性疾病和阿尔茨海默病以及重要的光。