Modeling Polycystic Kidney Disease Using Human Induced Pluripotent Stem Cells

描述(申请人提供)：常染色体显性遗传性多囊肾病(ADPKD)是人类最常见的威胁生命的遗传病，全世界有1：400到1：1000的人受到影响。ADPKD患者患有巨大的肾脏囊肿，最终导致肾功能衰竭，以及特殊的心血管问题，特别是可能破裂并导致死亡的颅内动脉瘤。ADPKD是由编码多囊蛋白-1和多囊蛋白-2的PKD1和PKD2的杂合性突变引起的，它们在肾上皮细胞和血管内皮细胞的初级纤毛上形成一个流量敏感通道复合体。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、更高通量的实验室模型来研究人类ADPKD。诱导多能干细胞(IPS)已经彻底改变了我们开发针对患者的体外疾病模型的能力。我们实验室最近从两名ADPKD患者(ADPKD-iPS)中培养了六个iPS细胞系。我们将首先证明这些是真正的多能iPS细胞系，能够从类胚体(EBS)的三层胚层中的每一层产生细胞类型。我们还将对这些细胞中的基因突变和多囊蛋白表达水平进行表征。为了将ADPKD-iPS细胞发展成体外疾病模型，ADPKD-iPS细胞或其衍生的EBS将被检测与睫毛功能或囊变相关的表型。我们将测量ADPKD-iPS或EB纤毛的形态，并与健康的iPS系进行比较，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将被分化为肾小管上皮细胞和内皮细胞，前者在体内形成ADPKD囊，后者可能导致颅内动脉瘤。这些细胞类型将被筛选出与上皮化和小管发生、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起着流动感受性钙通道的作用。最后，我们将使用siRNA多囊蛋白敲除来模拟“第二次命中”的体细胞突变来加剧表型，并使用野生型过表达来模拟基因治疗来挽救它们。建立ADPKD的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。

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.