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

使用报道人类 iPS 细胞研究命运、功能和帕金森病

• 批准号: 8841020
• 负责人: LORRAINE IACOVITTI
• 金额: $ 34.54万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8841020
• 关键词: 1-Methyl-4-phenylpyridinium; Adult; Affect; Astrocytes; Biochemical; Brain; Brain-Derived Neurotrophic Factor; Cell Culture Techniques; Cell Death; Cell Line; Cell Therapy; Cell model; Cell surface; Cells; Cues; DNA Sequence Alteration; Data; Dependovirus; Development; Disease; Dopamine; Embryo; Environment; Environmental Risk Factor; Epigenetic Process; Future; GDNF gene; Gene Proteins; Genetic; Genomics; Goals; Growth; Growth Factor; Heterogeneity; High Pressure Liquid Chromatography; Hip region structure; Human; Human Cell Line; In Vitro; Label; Midbrain structure; Mind; Modality; Modeling; Molecular Profiling; Mus; Neuroglia; Neurons; Neuroprotective Agents; Neurotoxins; Oligodendroglia; Oxidopamine; Parkinson Disease; Pathogenesis; Pathway interactions; Patients; Phenotype; Physiological; Pluripotent Stem Cells; Process; Production; Protocols documentation; Reporter; Role; Staging; Stem cells; Study models; System; Technology; Testing; Time; Toxin; Transgenes; Work; Zinc Fingers; brain cell; cell type; dopaminergic neuron; drug discovery; high throughput screening; human embryonic stem cell; immunocytochemistry; in vivo; induced pluripotent stem cell; injured; insight; integration site; nerve stem cell; neuron development; neurorestoration; neurotoxicity; novel; nuclease; progenitor; response; stem; success; tool; transcription factor; uptake

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.

描述（由申请人提供）：了解发育神经元中中脑多巴胺（mDA）表型分化的原理和过程不仅对于大脑个体发育很重要，而且对于帕金森病（PD）等疾病的研究和治疗也很重要。在过去的十年中，人们对胚胎小鼠大脑中调节 mDA 分化的转录机制有了深入的了解。重要的是，当人类诱导多能干 (hiPS) 细胞在培养皿中分化为 mDA 神经元时，许多相同的过程似乎是由它们共享的。因此，当源自人胚胎干 (hES) 细胞或成体诱导多能干 (hiPS) 细胞的人神经祖细胞 (hNP) 进入 mDA 分化途径时，它们表达许多相同的 mDA 特异性基因/蛋白质 (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%。 mDA 分化干细胞培养物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 mDA 细胞，对该领域产生了重大影响，阻碍了我们研究 mDA 分化机制或开发干细胞的能力作为体外帕金森病研究的模型或作为体内治疗方式。因此，在本提案中，我们的目标是使用锌指核酸酶将 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，创建新型报告 hiPS 干细胞系。这些荧光标记的细胞系将使我们能够在 mDA 分化过程中的不同阶段纯化细胞以达到同质性，并对控制 mDA 规范、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，我们将利用这些报告系和 DA 特异性神经毒素以及 PD 相关基因突变来开发 PD 干细胞模型，用于未来在培养中研究 PD 发病机制和潜在的 PD 治疗。