Using reporter human iPS cells to study fate, function and Parkinson's disease

描述(申请人提供)：了解支配发育中神经元的中脑多巴胺(MDA)表型分化的原理和过程不仅对大脑个体发育，而且对帕金森病(PD)等疾病的研究和治疗都很重要。在过去的十年里，人们对胚胎小鼠大脑中调控丙二醛分化的转录机制有了很大的了解。重要的是，当人类诱导的多能干细胞(HIPS)在培养皿中分化为丙二醛神经元时，许多相同的过程似乎都是相同的。因此，当来源于人类胚胎干细胞(HES)或成人诱导的多能干细胞(HIPS)的神经前体细胞(HNPs)致力于丙二醛(MDA)分化途径时，它们表达许多相同的丙二醛特异性基因/蛋白(Lmx1a、Aldh1a1、Nurr1、Pitx3、TH等)。重要的是，无论使用哪种分化方案，丙二醛神经元的最大产量很少超过总细胞的20%。丙二醛分化干细胞培养中细胞类型的异质性，加上目前缺乏合适的细胞表面标记来选择丙二醛细胞，已经严重影响了该领域，阻碍了我们研究丙二醛分化机制的能力，或者开发干细胞作为体外研究帕金森病的模型或作为体内治疗方式的能力。因此，在这项提案中，我们的目标是使用锌指核酸酶创建新的报告HIPS干细胞系，将GFP标记的MDA转基因插入到腺相关病毒(AAVS1)的安全港基因组整合位置。这些荧光标记的细胞系将使我们能够在丙二醛分化过程中的不同阶段将细胞纯化到同质性，并继续对控制丙二醛规格、中脑区域化和生理功能的遗传和表观遗传因素进行重要的概念验证研究。此外，我们将利用这些报道株和DA特异性神经毒素和PD相关基因突变来建立PD干细胞模型，为未来PD发病机制和潜在PD治疗的培养研究奠定基础。 公共卫生相关性：在这项提案中，我们将从正常人和帕金森患者中分离出人类诱导多能干细胞的报告细胞系。这些细胞将被提纯，并用于评估遗传、表观遗传和病理因素对多巴胺神经元发育和功能的影响。这项工作的总体目标是建立一个模型细胞系统，用于未来对中脑多巴胺分化的研究，对帕金森病(PD)的重要病理和环境机制的研究，为PD药物开发和最终开发基于细胞的治疗提供高通量筛选。

DESCRIPTION (provided by applicant): Understanding the principles and processes governing the differentiation of a midbrain dopamine (mDA) phenotype in developing neurons is important not only for brain ontogeny but also for the study and treatment of diseases such as Parkinson's disease (PD). In the last decade, a great deal of insight has been gained into the transcriptional machinery regulating mDA differentiation in the embryonic mouse brain. Importantly, many of those same processes appear to be shared by human induced pluripotent stem (hiPS) cells as they differentiate into mDA neurons in the dish. Thus, when human neural progenitors (hNPs) derived either from human embryonic stem (hES) cells or adult induced pluripotent stem (hiPS) cells commit to the mDA differentiation pathway, they express many of the same mDA-specific genes/proteins (Lmx1a, Aldh1a1, Nurr1, Pitx3, TH, etc.). Importantly, regardless of the differentiation protocol used, the maximum yield of mDA neurons rarely exceeds 20% of total cells. This heterogeneity of cell types in mDA-differentiated stem cell cultures combined with the current lack of suitable cell surface markers for the selection of mDA cells, has significantly impacted the field, hampering our ability to study the mechanisms underlying mDA differentiation or to develop stem cells as a model for the study of PD in vitro or as a treatment modality in vivo. Thus, in this proposal, our goal is to create novel reporter hiPS stem cell lines using zinc finger nucleases to insert GFP-tagged mDA transgenes into the adeno-associated virus (AAVS1) safe harbor genomic integration site. These fluorescently labeled cell lines will allow us to purify cells to homogeneity at distinct stages during the mDA differentiation process and proceed with important proof-of-concept studies on the genetic and epigenetic factors governing mDA specification, midbrain regionalization and physiological function. In addition, we will use these reporter lines and DA-specific neurotoxins and PD-related genetic mutations to develop a stem cell model of PD for future studies in culture on PD pathogenesis and potential PD treatments. PUBLIC HEALTH RELEVANCE: In this proposal, we will create reporter cells lines of human induced pluripotent stem cells isolated from normal subjects and Parkinson's patients. These cells will be purified and used in studies to assess the effects of genetic, epigenetic and pathological factors on dopamine neuron development and function. The overall goal of this work is to establish a model cell system for future studies on midbrain dopamine differentiation, for pathogenetic and environmental mechanisms important in Parkinson's disease (PD), for the production of a high throughput screen for PD drug discovery and ultimately for the development of cell-based therapies in PD.