Modeling Polycystic Kidney Disease Using Human Induced Pluripotent Stem Cells

使用人类诱导多能干细胞模拟多囊肾病

• 批准号: 8440919
• 负责人: Benjamin Solomon Freedman
• 金额: $ 5.84万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-06 至 2014-08-05
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8440919
• 关键词: Affect; Animal Model; Autosomal Dominant Polycystic Kidney; Blood Vessels; Calcium; Calcium Channel; Cardiovascular system; Cell Line; Cells; Cilia; Complex; Cyst; Defect; Development; Disease; Disease model; Endothelial Cells; Engineering; Epithelial; Epithelial Cells; Epithelium; Fluorescence; Functional disorder; Future; Gene Mutation; Germ Layers; Hereditary Disease; Human; Human Biology; Human Pathology; In Vitro; Individual; Intracranial Aneurysm; Kidney; Kidney Failure; Laboratories; Life; Light; Liquid substance; Measures; Modeling; Morphology; Mutation; PKD2 protein; Pathology; Patients; Phenotype; Pluripotent Stem Cells; Polycystic Kidney Diseases; Rupture; Sensory; Small Interfering RNA; Somatic Mutation; Testing; Therapeutic; Therapeutic Agents; Tissues; Tubular formation; Undifferentiated; Vascular Endothelial Cell; cell type; cilium biogenesis; disease phenotype; gene therapy; human disease; in vitro Model; in vitro testing; in vivo; induced pluripotent stem cell; molecular pathology; mortality; pluripotency; polycystic kidney disease 1 protein; research study; restoration; stem

DESCRIPTION (provided by applicant): Autosomal dominant polycystic kidney disease (ADPKD) is the most common life- threatening genetic disease in humans, affecting between 1:400 and 1:1000 people worldwide. ADPKD patients suffer from massive cysts in the kidney, leading to eventual renal failure, as well as specific cardiovascular problems, in particular intracranial aneurysms which can rupture and cause mortality. ADPKD is caused by heterozygous mutations in either PKD1 or PKD2 encoding polycystin-1 and polycystin-2, respectively, which form a flow-sensing channel complex at the primary cilium of renal epithelial and blood vessel endothelial cells. Since animals models are low-throughput and do not fully recapitulate human biology, more accurate and higher-throughput laboratory models are required to study human ADPKD. Induced pluripotent stem (iPS) cells have revolutionized our ability to develop patient-specific in vitro disease models. Our laboratory has recently generated six iPS cell lines from two ADPKD patients (ADPKD- iPS). We will first demonstrate that these are true pluripotent iPS cell lines capable of generating cell types from each of the three germ layers in embryoid bodies (EBs). We will also characterize the genetic mutations and polycystin expression levels in these cells. To develop ADPKD-iPS cells into an in vitro disease model, ADPKD-iPS cells or their derivative EBs will be inspected for phenotypes relating to ciliary function or cystogenesis. We will measure the morphology of ADPKD-iPS or EB cilia compared to healthy iPS lines and quantify ciliary polycystin levels by quantitative fluorescence intensity co-localization. In addition, ADPKD-iPS will be differentiated into either renal tubular epithelial cells, which form ADPKD cysts in vivo, or alternatively endothelial cells, which may give rise to intracranial aneurysms. These cell types will be screened for disease phenotypes related to epithelialization and tubulogenesis, ciliogenesis, and calcium release, since polycystins function as flow-sensory calcium channels at the primary cilium. Finally, we will model 'second hit' somatic mutations using siRNA polycystin knockdown to exacerbate phenotypes, and model gene therapy using wild- type over-expression to rescue them. Establishment of a human, in vitro model for ADPKD will enhance our understanding of disease pathology, allow for the testing of candidate therapeutic agents, and facilitate high-throughput therapeutics screens.

描述（由申请方提供）：常染色体显性遗传性多囊肾病（ADPKD）是人类最常见的危及生命的遗传性疾病，在全球范围内影响的患者比例为1：400至1：1000。ADPKD患者患有肾脏中的巨大囊肿，导致最终的肾衰竭，以及特定的心血管问题，特别是颅内动脉瘤，其可破裂并导致死亡。ADPKD是由分别编码多囊蛋白-1和多囊蛋白-2的PKD 1或PKD 2中的杂合突变引起的，所述杂合突变在肾上皮和血管内皮细胞的初级纤毛处形成流动传感通道复合物。由于动物模型的通量较低，并且不能完全概括人类生物学，因此需要更准确、更高通量的实验室模型来研究人类ADPKD。诱导多能干细胞（iPS）彻底改变了我们开发患者特异性体外疾病模型的能力。我们的实验室最近从两名ADPKD患者（ADPKD-iPS）中产生了六种iPS细胞系。我们将首先证明这些是真正的多能iPS细胞系，能够从拟胚体（EB）的三个胚层中的每一个产生细胞类型。我们还将表征这些细胞中的基因突变和多囊蛋白表达水平。为了将ADPKD-iPS细胞开发成体外疾病模型，将检查ADPKD-iPS细胞或其衍生EB与纤毛功能或囊肿发生相关的表型。我们将测量ADPKD-iPS或EB纤毛与健康iPS系相比的形态，并通过定量荧光强度共定位来定量纤毛多囊蛋白水平。此外，ADPKD-iPS将分化成肾小管上皮细胞，其在体内形成ADPKD囊肿，或者可替代地分化成内皮细胞，其可引起颅内动脉瘤。将筛选这些细胞类型的与上皮形成和小管形成、纤毛发生和钙释放相关的疾病表型，因为多囊蛋白在初级纤毛处起流动感觉钙通道的作用。最后，我们将使用siRNA多囊蛋白敲低来模拟“二次打击”体细胞突变以加剧表型，并使用野生型过表达来模拟基因治疗以拯救它们。建立人类ADPKD体外模型将增强我们对疾病病理学的理解，允许测试候选治疗药物，并促进高通量治疗筛选。