Modeling Pathogenesis of Huntington's disease using patient-derived induced pluri

描述（由申请人提供）：亨廷顿病（HD）是一种无法治愈的遗传性神经退行性疾病，由亨廷顿（Htt）基因外显子1中CAG重复序列的异常扩增引起。目前还不清楚为什么突变体Htt对神经元，特别是纹状体中的3-氨基丁酸（GABA）投射神经元的中型多刺神经元（MSN）有毒。人们普遍认为，缺乏对HD发病机制的了解的主要原因之一是缺乏可靠的实验模型。HD的细胞培养模型对于理解HD的分子机制是必不可少的，并且已被用于许多研究中。尽管先前开发的HD稳定细胞系表现出HD的一些病理特征，但它们几乎都不具有足够的神经元标记物或其他神经元特性，因此它们对于HD建模并不理想。为了更好地了解HD的发病机制，需要额外的新的HD细胞模型。最近在将人类体细胞重编程为诱导多能干（iPS）细胞方面的快速进展使得从HD患者产生iPS细胞模型成为可能。然而，由于目前的HD iPS细胞系含有用于递送几种重编程因子的多个病毒插入物，因此这些细胞是否能够很好地模拟HD是值得怀疑的，因为多个病毒插入物可能破坏内源性基因表达并引起异常细胞分化。为了克服这个问题，我们最近产生了来自HD患者成纤维细胞的无病毒和转基因iPS细胞，在这个项目中，我们建议使用这些iPS细胞来模拟HD。我们假设，这些HD iPS细胞，没有任何外源性DNA序列，可以成功地诱导分化为成熟的神经元，特别是GABA能神经元，一旦给予适当的条件。此外，我们假设，他们将重现HD的主要病理特征，在神经元成熟和老化的细胞培养。为了验证这些假设，提出了以下具体目标，其中两名本科生将参与研究。1.表征不含病毒或转基因插入物的HD患者源性iPS细胞系。2.诱导iPS细胞分化为GABA能神经元。3.目的：鉴定HD iPS细胞来源的GABA能神经元。PHS 398/2590（Rev. 11/07） 公共卫生相关性：亨廷顿氏病是一种遗传性神经系统疾病，选择性地导致特定脑区的神经细胞死亡，目前仍没有有效的治疗方法。尽管在过去的十年中已经建立了各种亨廷顿病的细胞和动物模型，但迄今为止还没有理想的模型可以复制这种疾病的所有基本病理特征。该项目旨在确定从患者诱导多能干细胞转化的神经细胞是否可以在细胞培养中模拟这种疾病的主要病理学方面。

DESCRIPTION (provided by applicant): Huntington's disease (HD) is an incurable inherited neurodegenerative disorder resulting from an abnormal expansion of CAG repeats in exon 1 of the huntingtin (Htt) gene. It remains unclear why mutant Htt is toxic to neurons, especially medium spiny neurons (MSNs) that are 3-aminobutyric acid (GABA) projection neurons in the striatum. It has been widely accepted that one of the major reasons for the lack of understanding of HD pathogenesis is the paucity of reliable experimental models. Cell culture models of HD are essential for understanding the molecular mechanisms of HD and have been employed in a number of studies. Although the HD stable cell lines developed previously exhibit some of the pathological features of HD, almost none of them possess adequate neuronal markers or other neuronal properties and thus they are not ideal for modeling HD. To better understand the pathogenesis of HD, additional novel cell models of HD are required. Recent rapid advances in reprogramming human somatic cells into induced pluripotent stem (iPS) cells have made it possible to generate iPS cell models from HD patients. However, since the current HD iPS cell lines contain multiple viral inserts that are used for delivering several reprogramming factors, it is questionable whether these cells can model HD well because the multiple viral inserts may disrupt endogenous gene expression and cause abnormal cellular differentiation. To overcome this problem, we recently generated virus- and transgene-free iPS cells derived from HD patient fibroblasts and, in this project, we propose to model HD using these iPS cells. We hypothesize that these HD iPS cells, free of any exogenous DNA sequence, can be successfully induced to differentiate into mature neurons, especially GABAergic neurons, once adequate conditions are given. Additionally, we hypothesize that they will reproduce the major pathological features of HD across neuronal maturation and aging in cell culture. To test these hypotheses the following specific aims are proposed, in which two undergraduate students will participate in the studies. 1. To characterize HD patient-derived iPS cell lines free of viral or transgenic inserts. 2. To induce the iPS cells to differentiate into GABAergic neurons. 3. To characterize the GABAergic neurons derived from the HD iPS cells. PHS 398/2590 (Rev. 11/07) Page Continuation Format Page PUBLIC HEALTH RELEVANCE: Huntington's disease is an inherited neurological disorder that selectively causes nerve cells to die in the specific brain regions and effective therapy for this disease is still unavailable at present. Although various cell and animal models of Huntington's disease have been created over last decade, so far there is no ideal model that can replicate all of essential pathological features of this disease. This project is designed to determine whether the nerve cells that are converted from patient induced pluripotent stem cells can mimic major aspects of pathology of this disorder in cell culture.