Development of a fluidic chip model of PKD to elucidate cystogenic signals using kidney organoids

项目摘要 来源于人多能干细胞（hPSC）的肾脏类器官可以成为研究肾脏的新工具 疾病多囊肾病（PKD）是最常见的遗传性肾病， 终末期肾病，困扰着全世界超过1200万人。动物和静态肾类器官模型 已经被开发来理解用于治疗开发的囊肿形成的病理机制。 然而，ARPKD小鼠的肾脏表型通常不存在，当存在时，它是一种轻度病变， 涉及近端小管而不是远端肾单位。相比之下，人类肾脏类器官模型可以提供 PKD发病机制的新见解，但目前的ARPKD模型是用化学诱导剂开发的， 毛喉素，在静态培养，表现出近端小管囊肿。 最近，我们的合作团队开发了一种新的芯片上类器官模型， 剪切应力可施加于整个肾类器官。有趣的是，我们的初步结果使用流体 芯片显示临床相关的表型，在培养条件下的PKHD 1-/-类器官中具有远端肾单位扩张。 流这些新的工具结合了肾脏类器官，芯片上的肾脏和CRISPR基因组编辑可能会提供 有效探索参与囊肿形成和信号的信号途径的新机会 这些分子可以成为ARPKD患者的新治疗靶点。 为了开发ARPKD的生理模型，在具体目标1中，我们将从 PKHD 1-突变型hPSC。然后，我们将优化微流控芯片上的培养条件， 体外肾类器官。流动引起的机械应力，包括纤毛应力和细胞拉伸，将被 通过ELISA、生物传感器和转录组学进行评估。我们将验证流体ARPKD模型通过比较 囊性表型与人ARPKD肾脏样品的比较。对于机制研究，在具体目标2中，我们将 生成额外的工具：1.在PKHD 1-/-hPSC中通过CRISPR基因组编辑的初级纤毛敲除系，和2. 使用人SSTR 3-GFP的原代纤毛报告细胞系。纤毛敲除系将使机制研究成为可能 涉及ARPKD肾和其他类器官中纤毛应激介导的致病信号。记者线 将能够在各种实验环境中对初级纤毛运动进行实时成像， 移植动物中的类器官芯片和活体成像。 我们提出的工作将为PKD和纤毛病研究社区产生新的体外工具， 探讨纤毛应激诱导肾脏及其它类器官囊肿形成的病理机制。 来自灌注的肾类器官的转录组学数据也将提供对膀胱发生的机制性见解。 ARPKD。本研究开发的所有工具和数据将通过NIH PKD和RBK传播 联合体，以促进PKD患者的治疗开发。

Project Summary Kidney organoids derived from human pluripotent stem cells (hPSCs) can be a novel tool to study kidney diseases. Polycystic kidney disease (PKD) is the most common genetic kidney disease which results in end-stage kidney disease, afflicting over 12 million people worldwide. Animal and static kidney organoid models have been developed to understand the pathomechanisms of cystogenesis for therapeutic development. However, renal phenotypes in ARPKD mice are often absent, and when present, it is a mild lesion that typically involves proximal tubules rather than distal nephrons. In contrast, human kidney organoid models could provide new insights into PKD pathogenesis, yet current ARPKD models are developed with a chemical inducer, forskolin, in static culture, exhibiting proximal tubular cysts. Recently, our collaborative team has developed a new model of organoid-on-the-chip by which fluidic shear stress can be applied to whole kidney organoids. Interestingly, our preliminary results using the fluidic chips showed clinically relevant phenotypes with distal nephron dilatation in PKHD1-/- organoids cultured under flow. These novel tools marrying kidney organoid, kidney-on-a-chip, and CRISPR genome editing might offer new opportunities to efficiently explore the signal pathways that are involved in cyst formation and signal molecules that can be new therapeutic targets for ARPKD patients. To develop a physiologic model of ARPKD, in Specific Aim 1, we will generate kidney organoids from PKHD1-mutant hPSCs. Then we will optimize culture conditions on millifluidic chips to apply shear stress to kidney organoids in vitro. Flow-induced mechanical stress including ciliary stress and cellular stretching will be evaluated by ELISA, biosensors, and transcriptomics. We will validate the fluidic ARPKD model by comparing cystic phenotypes to human ARPKD kidney samples. For mechanistic studies, in Specific Aim 2, we will generate additional tools: 1. primary cilia knock-out lines by CRISPR genome editing in PKHD1-/- hPSCs, and 2. primary cilia reporter lines using human SSTR3-GFP. Cilia knock-out lines will enable mechanistic studies involving ciliary stress-mediated pathogenic signals in ARPKD kidney and other organoids. The reporter lines will enable real-time imaging of primary cilia movement in varied experimental settings including organoid-on-chip and intravital imaging in transplanted animals. Our proposed work will generate novel in vitro tools for PKD and ciliopathy research communities to investigate pathomechanisms of cystogenesis induced by ciliary stress in kidney and other organoids. Transcriptomic data from perfused kidney organoids will also provide mechanistic insights into cystogenesis in ARPKD. All tools and data developed by this study will be disseminated through NIH PKD and RBK consortiums to facilitate therapeutic development for PKD patients.