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）： 该提案研究了使用诱导多能干细胞 (iPSC) 衍生的内皮细胞 (EC) 作为天然 EC 的替代物，以增进我们的理解并更好地治疗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，他们在 PAH 的临床表现和病理学、iPSC 生成和分化、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体技术方面拥有专业知识。我们的提案反映了评估来自同一患者的三种 EC 系的独特机会。这简化了我们测试 iPSC-EC 作为天然 EC 替代物的功效的能力，并确定特定基因改变的重要性。我们有一个独特的机会来比较来自相同 PAH 患者或对照的成纤维细胞衍生的 iPSC-EC 与肺动脉 (PA)EC 衍生的 iPSC-EC 的基因变异、表观遗传和基因表达谱。这应该以前所未有的方式解决遗传脆弱性如何导致明显疾病的问题。此外，我们有能力研究基因和药物治疗在 iPSC-EC 中逆转 PAH 病理学的应用。由于 Wu 实验室迄今为止完成的研究已经建立了 iPSC-EC 的生产设施，因此我们准备从本提案的第二阶段开始。为此，我们提出第二阶段的三个具体目标和第三阶段的三个具体目标。第二阶段目标 1 应用 Hi-Seq、Mmethyl-Seq 和 RNA-Seq 来获取 PAH 和对照成纤维细胞 - iPSC-EC、PAEC-iPSC-EC 和 PAEC 中罕见基因变异、表观遗传变化和基因表达的信息。第二阶段的目标 2 通过使用血管生成测定和磷流分析对这些细胞的表型进行广泛分析，并使用单细胞微流体方法评估同质性，将基因组信息与功能相关联。 II 期 Aim 3 启动与 Progenitor Cell Therapy 的合作，转让 iPSC-EC 生产技术，以便未来能够扩大规模，造福大量患者。在 III 期中，目标 1 利用尖端基因治疗方法（例如小环和核糖体 DNA 载体）来纠正 iPSC-EC 中的基因变异。 第三阶段，目标 2 研究如何恢复细胞的 PAH 相关表型。 III 期目标 3 比较了成纤维细胞和 PAEC-iPSC-EC 与天然 PAEC 响应药物恢复疾病表型的功效。总之，我们的研究应该作为一个模型，更好地了解如何使用 iPSC-EC 来发现和治疗多种心血管疾病中血管功能障碍的遗传倾向。 （摘要完）