Human Stem Cell Model of Niemann Pick Type C

描述(申请人提供)：Niemann Pick C型(NPC1)是一种罕见但致命的儿童痴呆症，由NPC1突变引起，NPC1是一种存在于晚期内膜间室的管家蛋白，可能在胆固醇运输中发挥作用。其结果是一种严重的脂肪症，其特征是大量积累溶酶体、类固醇和其他类脂，最终导致细胞死亡。这种疾病具有巨大的基础科学价值，也是细胞内胆固醇运输障碍的标志性模型，因为它与阿尔茨海默病(AD)相似，后者表明了共同的潜在机制。到目前为止，建立鼻咽癌模型的策略尚未确定NPC1的某些突变如何导致人类神经元功能衰竭，原因有两个：i)使用不能复制人类病理的所有方面的动物模型；ii)关注胆固醇的积累是鼻咽癌神经元功能障碍的原因，这可能不是神经元细胞群中的主要表型。我们建议通过对人类胚胎干细胞(HESCs)进行基因工程并将体细胞重新编程为人诱导多能干细胞(HiPSCs)来生成第一个NPC1的人类神经元模型。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HiPSCs研究神经退行性疾病的研究。通过对hESC和hiPSC的平行分析，我们还将解决鼻咽癌的遗传异质性，并确认在这些细胞中发现的病理表型是由于缺乏正常的NPC1功能所致。我们将利用shRNA介导的NPC1沉默和病毒重编程的插入方法，分别获得NPC1基因敲除的hESC和鼻咽癌成纤维细胞重编程的HiPSC。我们将严格遵循这些新产生的株系的特征标准，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。我们将使用在我的实验室开发的方案来生成纯人类神经元群体，我们将在批量、纯和隔室培养中进行研究。我们将分析来自NPC hESCs和hiPSCs的人类神经元，以测试在人类神经元疾病模型中从未探索过的特定预测；i)我们将确认并扩大尚未在活体人类神经元中测试的NPC典型表型；ii)我们将评估野生型和NPC1神经元的生存和分化能力；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；iv)我们将探索NPC是否是一种细胞自主疾病，或者NPC中的神经元衰竭是否会受到胶质环境的影响。从我们的观察中产生的见解有可能极大地提高我们对NPC1如何导致人类神经元衰竭的理解，并指导可能导致治愈方法的开发工作。加州大学圣迭戈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，UCSD在2007财年的研发支出近8亿美元。这个项目将为我们的机构增加一个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。未来的经济安全将通过继续雇用一名高级内科科学家、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创造知识，培养下一代学术劳动力，开发新技术，为未来的研究提供机会，并在学术部门利用有可能创造更多就业机会的商业服务。 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建立第一个人类Niemann Pick C1型神经元模型。我们将使用对照和NPC1人类神经元来研究胆固醇在NPC1中的运输动力学，以及胆固醇在神经元存活和再生中的作用。这项工作将为一种重要的儿童神经退行性疾病和阿尔茨海默病提供重要的线索。

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.