Generation and Characterization of Amyotrophic Lateral Sclerosis

来自ALS患者的诱导多能细胞(IPS)可以为我们提供一个特殊的工具，用来研究疾病的病理生理和可用药的靶点。最近对肌萎缩侧索硬化症的病理生理学的许多见解来自于对这种疾病的家族性形式的研究。实际拥有人类细胞系的能力-在最相关的细胞类型-运动神经元和星形胶质细胞中代表自然疾病-提供了前所未有的工具：1)研究导致疾病病理生理学的细胞相互作用；2)为药物发现和遗传路径分析提供关键工具。我们在相同的条件下，从各种家族性肌萎缩侧索硬化症(FAL)突变和散发性肌萎缩侧索硬化症(SALS)患者中建立了iPS细胞系，以确定它们在细胞类型特异性和功能生物学方面的代表性。现在产生的第一组细胞已经准备好用于疾病表型范例。整个提案将涉及四名主要研究人员，他们密切合作，生成和评估FALS和SALS细胞系，并利用它们开发细胞特异性表型分析。由Eggan博士领导的项目1将生成新的关键IPS系--从选定的FAL突变以及非整合IPS系中产生等基因留置权--以补充我们基于逆转录病毒的IPS收集。此外，项目1将使用一种新的遗传记分卡系统，全面评估这些品系的多能性。具有神经/神经胶质特征的iPS细胞株将被送往由克里斯·亨德森领导的项目2实验室-运动神经元生物学实验室和项目3实验室。星形细胞--由杰弗里·罗斯坦领导。这两个项目/实验室将通过序贯分析确定哪些FALS IPS细胞系具有适当的运动神经元和星形胶质细胞特征。此外，这两个小组都将产生基于锌手指的细胞特异性报告细胞系，用于未来的药物发现分析。符合最终标准的细胞系(与人类ES细胞和之前对人类星形胶质细胞的研究相比)将在Tom Maniatis领导的Project 4实验室进行基因分析。最后，为了开发治疗和发病机制的有用工具，核心1和2将产生运动神经元和星形胶质疾病的表型分析。

Induced Pluripotent Cells (IPS) from ALS patients could provide an exceptional tool by which we can Disease pathophysiology and druggable targets. Many recent insights into the pathophysiology of ALS come from the study of familial forms of this disease. The ability to actually have human cell lines-representing the natural disease in the most relevant cell types- motor neurons and astrocytes- provides unprecedented tools to 1) study cell interactions responsible for disease pathophysiology and 2) provide critical tools for drug discovery and genetic pathway analysis. We have generated iPS cell lines, under identical conditions, from various familial ALS (fALS) mutations and sporadic ALS (sALS) patients to determine how representative they are for cell type specificity and functional biology. The first set of cells now generated are ready for use in disease phenotyping paradigms. The overall proposal will involve four principal investigators, working in tight collaboration, to generate and evaluate fALS and sALS cell lines and to use them to develop cell specific phenotypic assays. Project 1, lead by Dr. Eggan will generate new critical IPS line- isogenic liens from selected fALS mutations as well as non-integrating IPS lines- to complement our retroviral based IPS collection. In addition Project 1 will fully evaluate the pluripotency of the lines using a novel genetic scorecard system. iPS cell lines with neural/glial characteristics will be sent to the Project 2 Lab- Motor neuron biology, lead by Chris Henderson and to Project 3 lab. Astrocytes- lead by Jeffrey Rothstein. These two projects/labs will determine which of the fALS IPS cell lines have the appropriate characteristics of motor neurons and astroglia, thru sequential analyses. In addition, both groups will generate Zinc-finger based cell specific reporter cell lines for future use in drug discovery assays. Cell lines that meet final criteria (as compared to human ES cell and prior work on human astroglia) will undergo genetic analysis in the Project 4 lab, lead by Tom Maniatis. Finally, to develop useful tools for therapeutics and pathogenesis, Cores 1 and 2 will generate motor neuron and astroglial disease phenotyping assays.