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: 8297223
• 负责人: LORRAINE IACOVITTI
• 金额: $ 38.01万
• 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 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8297223
• 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; Commit; Cues; Data; Dependovirus; Development; Disease; Dopamine; Embryo; Environment; Environmental Risk Factor; Epigenetic Process; Future; GDNF gene; Gene Mutation; 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; Site; 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; 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. 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.

描述（由申请人提供）：了解开发神经元中中脑多巴胺（MDA）表型分化的原理和过程不仅对脑本体发育，而且对诸如帕金森病（PD）等疾病的研究和治疗也很重要。在过去的十年中，已经在调节胚胎小鼠大脑中MDA分化的转录机制中获得了很多见解。重要的是，许多相同的过程似乎是由人类诱导的多能茎（臀部）细胞分化为菜肴中的MDA神经元时共享的。因此，当人类神经祖细胞（HNP）源自人类胚胎（HES）细胞或成年诱导的多能干（臀部）细胞的诱导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，lmx1a，aldh1a1，aldh1a1，nurr1，pitx3，pitx3，th，th，th，等）。重要的是，不管使用的分化方案如何，MDA神经元的最大产量均未超过总细胞的20％。在MDA分差的干细胞培养物中，细胞类型的这种异质性结合了当前缺乏用于选择MDA细胞的合适细胞表面标记，已显着影响该领域，这阻碍了我们研究MDA分化基础机制或发展为PD在VERRO或治疗模态的模型的干细胞中的机制的能力。因此，在此提案中，我们的目标是使用锌指核酸酶创建新的记者臀部干细胞系，以将GFP标记的MDA转基因插入与腺相关病毒（AAVS1）安全港基因组整合位点中。这些荧光标记的细胞系将使我们能够在MDA分化过程中在不同阶段净化细胞，并进行有关管理MDA规范，中脑区域化和生理功能的遗传和表观遗传因素的重要概念证明研究。此外，我们将使用这些报告基因线和DA特异性神经毒素和与PD相关的遗传突变来开发PD的干细胞模型，以在PD发病机理和潜在PD治疗的培养中进行未来的研究。 公共卫生相关性：在此提案中，我们将创建从正常受试者和帕金森患者分离的人类诱导多能干细胞的报告细胞系列。这些细胞将被纯化并用于研究，以评估遗传，表观遗传学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，用于对中脑多巴胺分化的未来研究，对于帕金森氏病（PD）重要的致病和环境机制，以生产PD药物发现的高吞吐量筛查，并最终用于开发PD中基于细胞的疗法。