Human Stem Cell Model of Niemann Pick Type C

Niemann Pick C型人类干细胞模型

基本信息

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
项目类别：
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&apos 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

项目摘要

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 C型（NPC1）是由NPC1突变引起的一种罕见但致命的小儿痴呆，这是一种居住在晚期内体内室中的管家蛋白，在胆固醇转运中起假定作用。结果是严重的脂质病，其特征是溶酶体固醇和其他最终导致细胞死亡的脂质的大量积累。该疾病具有巨大的基础科学兴趣以及功能失调的细胞内胆固醇运输的标志性模型，并且由于它与阿尔茨海默氏病（AD）的相似之处，该模型暗示了共同的潜在机制。 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.我们建议通过人类胚胎干细胞（HESC）的基因工程以及将体细胞重编程为人类诱导的多能干细胞（HIPSC）来生成NPC1的第一个人类神经元模型。从广义的角度来看，我们的方法将在干细胞场中产生重大影响，因为它交叉验证了HIPSC的研究研究神经退行性疾病。通过对HESC和HIPSC线进行并行分析，我们还将解决NPC的遗传异质性，并确认在这些细胞中发现的病理表型特别是由于缺乏正常的NPC1功能。我们将使用shRNA介导的NPC1的沉默和病毒重编程的插入方法来生成独立的NPC1敲低hESC线的集合，并分别从NPC成纤维细胞重新编程的HIPSC线。我们将遵循这些新生成的线的严格表征标准，以确保它们保持干细胞特性，在遗传上是稳定的，并复制了小鼠文献中描述的基本NPC表型。我们将使用我们在实验室中开发的方案来产生我们将以散装，纯净和隔室文化研究的纯人类神经元的种群。我们将分析源自NPC HESC和HIPSC的人神经元，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。 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或者，如果NPC中的神经元衰竭可能受神经胶质环境的影响。从我们的观察结果中产生的见解有可能大大提高我们对NPC1如何导致人类神经元失败的理解，并指导努力可能导致治疗的发展。 UCSD是该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8亿美元的研发。该项目将在我们的机构中成为州和国家一级的经济和学术引擎。我们的项目将通过研究，创新和创造就业机会为地区和国民经济的持续发展做出贡献。未来的经济安全将通过一位高级研究技术员以及研究生和本科生的高级医师科学家的持续就业来促进未来的经济安全。这项投资的长期影响将是创造知识，下一代学术劳动力的准备，新技术的发展，未来研究的机会以及在学术领域使用商业服务的机会，这些商业服务有可能产生更多的工作。公共卫生相关性：我们建议从人类胚胎干细胞和人类诱导的多能干细胞中创建第一个人类神经元模型。我们将使用控制和NPC1人神经元来研究NPC1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。这项工作将对重要的童年神经退行性疾病和阿尔茨海默氏病的重要启示。