Modeling Polycystic Kidney Disease Using Human Induced Pluripotent Stem Cells

描述（由申请人提供）：常染色体显性多囊肾病（ADPKD）是人类最常见的威胁生命的遗传性疾病，在世界范围内影响1:400至1:1000的人群。ADPKD患者会在肾脏中出现巨大的囊肿，导致最终的肾功能衰竭，以及特定的心血管问题，特别是颅内动脉瘤，可能会破裂并导致死亡。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，多囊蛋白-1和多囊蛋白-2在肾上皮细胞和血管内皮细胞的初级纤毛处形成一个流量感应通道复合物。由于动物模型是低通量的，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患者身上获得了6个iPS细胞系（ADPKD- iPS）。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（EBs）的三个胚层中的每一个产生细胞类型。我们还将描述这些细胞中的基因突变和多囊蛋白表达水平。为了将ADPKD-iPS细胞培养成体外疾病模型，将检查ADPKD-iPS细胞或其衍生物EBs的纤毛功能或膀胱发生相关表型。我们将测量ADPKD-iPS或EB纤毛的形态，与健康iPS系进行比较，并通过定量荧光强度共定位定量纤毛多囊素水平。此外，ADPKD- ips会分化为肾小管上皮细胞，在体内形成ADPKD囊肿，或者分化为内皮细胞，可能导致颅内动脉瘤。这些细胞类型将筛选与上皮化、小管形成、纤毛形成和钙释放相关的疾病表型，因为多囊素在初级纤毛上起着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来拯救它们。建立人类ADPKD体外模型将增强我们对疾病病理学的理解，允许候选治疗药物的测试，并促进高通量治疗筛选。

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.