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

基本信息

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
项目类别：
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

项目摘要

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（由申请人提供）：常染色体显性多囊肾病 (ADPKD) 是人类最常见的危及生命的遗传病，影响全世界 1:400 至 1:1000 人。 ADPKD 患者患有肾脏巨大囊肿，最终导致肾衰竭，以及特定的心血管问题，特别是颅内动脉瘤，可能破裂并导致死亡。 ADPKD 是由分别编码多囊蛋白-1 和多囊蛋白-2 的 PKD1 或 PKD2 杂合突变引起的，它们在肾上皮细胞和血管内皮细胞的初级纤毛处形成血流传感通道复合物。由于动物模型的通量低且不能完全概括人类生物学，因此需要更准确和更高通量的实验室模型来研究人类 ADPKD。诱导多能干 (iPS) 细胞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 6 种 iPS 细胞系。我们将首先证明这些是真正的多能 iPS 细胞系，能够从胚状体 (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 的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量化纤毛多囊蛋白水平。此外，ADPKD-iPS将分化为肾小管上皮细胞（在体内形成ADPKD囊肿）或内皮细胞（可能引起颅内动脉瘤）。这些细胞类型将被筛选与上皮化和管状发生、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充当流感钙通道。最后，我们将使用 siRNA 多囊蛋白敲低来模拟“第二次打击”体细胞突变，以加剧表型，并使用野生型过度表达来模拟基因治疗来拯救它们。建立 ADPKD 人体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。