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，人们对小鼠胚胎大脑中调节mDA分化的转录机制有了大量的了解。重要的是，当人类诱导多能干细胞（hiPS）在培养皿中分化为mDA神经元时，许多相同的过程似乎与它们相同。因此，当来自人胚胎干细胞（hES）细胞或成人诱导多能干细胞（hiPS）细胞的人类神经祖细胞（hNPs）参与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，无论采用何种分化方案，mDA神经元的最大产量很少超过总细胞的20%。mDA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物来选择mDA细胞，对该领域产生了重大影响，阻碍了我们研究mDA分化机制的能力，也阻碍了我们开发干细胞作为PD体外研究模型或体内治疗方式的能力。因此，在这项提议中，我们的目标是利用锌指核酸酶将gfp标记的mDA转基因插入腺相关病毒（AAVS1）安全港基因组整合位点，从而创建新的报告细胞hiPS干细胞系。这些荧光标记的细胞系将使我们能够在mDA分化过程的不同阶段纯化细胞，并进行重要的概念验证研究，研究控制mDA规格、中脑区域划分和生理功能的遗传和表观遗传因素。此外，我们将利用这些报告系和da特异性神经毒素以及PD相关基因突变来建立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。

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.