iPSC Derived EC as Surrogates Using Pulmonary Hypertension as a Prototype Disease

使用肺动脉高压作为原型疾病的 iPSC 衍生 EC 作为替代物

• 批准号: 8689146
• 负责人: Marlene Rabinovitch
• 金额: $ 229.01万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-05 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8689146
• 关键词: Address; Bioinformatics; Biological Assay; Cardiovascular Diseases; Cell Therapy; Cells; Clinical; Disease; Endothelial Cells; Epigenetic Process; Fibroblasts; Future; Gene Expression; Generations; Genes; Genetic Drift; Genetic Predisposition to Disease; Genomics; Laboratories; Life; Lung; Microfluidics; Modeling; Molecular Profiling; Mutation; Pathology; Patients; Pharmacologic Substance; Phase; Phenotype; Positioning Attribute; Production; Pulmonary Hypertension; Ramp; Ribosomal DNA; Stem cells; Technology; Technology Transfer; Vascular Diseases; abstracting; angiogenesis; comparative efficacy; disease phenotype; efficacy testing; gene therapy; genetic variant; hypertension control; improved; induced pluripotent stem cell; multidisciplinary; patient population; prototype; transcriptome sequencing; vector

DESCRIPTION (provided by applicant): This proposal investigates the use of induced pluripotent stem cell (iPSC)-derived endothelial cells (ECs) as surrogates for native ECs to improve our understanding and to better treat a life-threatening vascular disorder, pulmonary hypertension (PAH). To this end we have assembled a multidisciplinary team with expertise in clinical manifestations and pathobiology of PAH, iPSC generation and differentiation, genetics and genomics, bioinformatics and gene therapy and high throughput phosphoflow and microfluidic technologies. Our proposal reflects a unique opportunity to assess three lines of ECs derived from the same patient. This streamlines our ability to test the efficacy of iPSC-ECs as surrogates for native ECs, and to establish the significance of specific genetic alterations. We have a unique opportunity to compare gene variants, epigenetic and gene expression profiles in fibroblast derived iPSC-ECs to those in pulmonary arterial (PA)EC derived iPSC-ECs from the same PAH patients or controls. This should address in an unprecedented manner, how a genetic vulnerability leads to manifest disease. Moreover, we are well placed to investigate the application of gene and pharmaceutical therapy to reverse PAH pathology in iPSC-ECs. Because studies completed to date in the Wu laboratory have established facility with the production of iPSC-ECs, we are positioned to begin with Phase II of this Proposal. To this end, we propose three Specific Aims in Phase II and three in Phase III. Phase II, Aim 1 applies Hi-Seq, Methyl-Seq and RNA-Seq to obtain information on rare gene variants, epigenetic changes and gene expression in PAH and control fibroblast- iPSC-ECs, PAEC-iPSC-ECs and PAECs. Phase II, Aim 2 correlates this genomic information with function, by extensive analysis of the phenotype of these cells using angiogenesis assays and phosphoflow analyses and by assessing homogeneity using a single cell microfluidic approach. Phase II, Aim 3 initiates a partnership with Progenitor Cell Therapy to transfer the technology of producing iPSC-ECs so that it can be ramped up in the future to benefit large populations of patients. In Phase III, Aim 1 utilizes cutting edge gene therapy approaches, such as minicircles and ribosomal DNA vectors, to correct a gene variant in an iPSC-EC. Phase III, Aim 2 investigates how this reverts the PAH-related phenotype of the cell. Phase III, Aim 3 compares the efficacy of fibroblast and PAEC-iPSC-ECs vs. native PAECs to respond to pharmaceuticals to revert the disease phenotype. In summary, our studies should serve as a model to better understand how iPSC-ECs can be used to uncover and treat genetic predisposition to vascular dysfunction in a wide variety of cardiovascular diseases. (End of Abstract)

描述(由申请人提供)： 这项建议研究使用诱导多能干细胞(IPSC)来源的内皮细胞(ECs)作为天然ECs的替代品，以提高我们的认识，并更好地治疗一种危及生命的血管疾病--肺动脉高压(PAH)。为此，我们组建了一支多学科团队，在PAH的临床表现和病理生物学、IPSC的产生和分化、遗传学和基因组学、生物信息学和基因治疗以及高通量磷流和微流控技术方面拥有专业知识。我们的建议反映了一个独特的机会，可以评估来自同一患者的三种ECs。这使我们能够测试IPSC-ECs作为本地ECs替代品的有效性，并确定特定基因改变的意义。我们有一个独特的机会来比较成纤维细胞来源的IPSC-ECs和来自相同PAH患者或对照的肺动脉(PA)EC来源的IPSC-ECs的基因变异、表观遗传学和基因表达谱。这应该以前所未有的方式解决遗传脆弱性如何导致明显的疾病。此外，我们很好地研究了基因和药物治疗在逆转IPSC-ECs PAH病理上的应用。由于到目前为止在吴实验室完成的研究已经建立了生产IPSC-ECs的设施，我们已经准备好从这项提议的第二阶段开始。为此，我们在第二阶段提出了三个特定的目标，并在第三阶段提出了三个目标。第二阶段，目标1应用Hi-Seq、甲基-Seq和RNA-Seq来获得关于PAH中稀有基因变异、表观遗传变化和基因表达的信息，并控制成纤维细胞-IPSC-ECs、PAEC-IPSC-ECs和PAECs。第二阶段，AIM 2通过使用血管生成分析和磷流分析对这些细胞的表型进行广泛分析，并通过使用单细胞微流控方法评估同质性，将这些基因组信息与功能相关联。第二阶段，AIM 3启动了与祖细胞疗法的合作伙伴关系，以转让生产IPSC-ECs的技术，以便在未来能够扩大到造福于大量患者。在第三阶段，AIM 1利用尖端基因治疗方法，如微环和核糖体DNA载体，纠正IPSC-EC中的基因变异。第三阶段，AIM 2研究这如何逆转细胞的多环芳烃相关表型。第三阶段，目的3比较成纤维细胞和PAEC-IPSC-ECs与天然PAEC对药物逆转疾病表型的疗效。总之，我们的研究应该作为一个模型，以更好地了解IPSC-ECs如何用于揭示和治疗各种心血管疾病中血管功能障碍的遗传易感性。 (摘要结束)