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

描述（由申请人提供）：了解发育中神经元中脑多巴胺（mDA）表型分化的原理和过程不仅对脑个体发育很重要，而且对帕金森病（PD）等疾病的研究和治疗也很重要。在过去的十年中，已经获得了大量的洞察到胚胎小鼠脑中的mDA分化的转录调节机制。重要的是，许多相同的过程似乎是由人类诱导多能干细胞（hiPS）共享的，因为它们在培养皿中分化为mDA神经元。因此，当源自人胚胎干（hES）细胞或成人诱导多能干（hiPS）细胞的人神经祖细胞（hNP）致力于mDA分化途径时，它们表达许多相同的mDA特异性基因/蛋白（Lmx1a、Aldh1a1、Nurr1、Pitx3、TH等）。重要的是，无论使用何种分化方案，mDA神经元的最大产量很少超过总细胞的20%。在mDA分化的干细胞培养物中细胞类型的这种异质性与目前缺乏用于选择mDA细胞的合适的细胞表面标志物相结合，显著影响了该领域，阻碍了我们研究mDA分化的机制或开发干细胞作为体外PD研究模型或作为体内治疗方式的能力。因此，在这个建议中，我们的目标是创建新的报告hiPS干细胞系，使用锌指核酸酶插入GFP标记的mDA转基因到腺相关病毒（AAVS1）的安全港基因组整合位点。这些荧光标记的细胞系将使我们能够在mDA分化过程中的不同阶段将细胞纯化至均一性，并对控制mDA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，我们将使用这些报告细胞系和DA特异性神经毒素和PD相关的基因突变来开发PD的干细胞模型，用于未来PD发病机制和潜在PD治疗的培养研究。 公共卫生相关性：在这个提议中，我们将创建从正常受试者和帕金森病患者中分离的人类诱导多能干细胞的报告细胞系。这些细胞将被纯化并用于研究，以评估遗传，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，用于未来研究中脑多巴胺分化，帕金森病（PD）的重要发病机制和环境机制，用于PD药物发现的高通量筛选的生产，并最终用于PD中基于细胞的治疗方法的开发。

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.