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

项目摘要 人多能干细胞(HPSCs)来源的肾脏器官可作为肾脏研究的新工具 疾病。多囊肾病(PKD)是最常见的遗传性肾脏疾病，可导致 终末期肾病，困扰着全世界1200多万人。动物和静态肾脏器官模型 已被用来了解膀胱发生的病理机制，以用于治疗开发。 然而，ARPKD小鼠的肾脏表型通常是缺失的，当存在时，这是一种轻微的病变，通常 累及近端肾小管而不是远端肾单位。相比之下，人类肾脏器官模型可以提供 对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， Forskolin，在静态培养中，表现为近端管状囊肿。 最近，我们合作的团队开发了一种新的芯片上有机化合物模型，通过这种模型，流体 切应力可以应用于整个肾脏器官。有趣的是，我们的初步结果使用了流体力学 在体外培养的PKHD1-/-类器官中，CHIP显示出与远端肾单位扩张相关的临床表型 流。这些结合肾脏器官、芯片上肾脏和CRISPR基因组编辑的新工具可能会提供 有效探索与包囊形成和信号传导有关的信号通路的新机会 可以成为ARPKD患者新的治疗靶点的分子。 为了建立ARPKD的生理模型，在特定的目标1中，我们将从 PKHD1-突变体hPSCs。然后，我们将优化微流控芯片的培养条件，以施加剪应力 肾脏有机化合物的体外实验。流体诱导的机械应力，包括纤毛应力和细胞拉伸 通过酶联免疫吸附试验、生物传感器和转录组分学进行评估。我们将通过比较来验证流体ARPKD模型 人类ARPKD肾脏样本的囊性表型。对于机械论研究，在具体目标2中，我们将 生成其他工具：1.在PKHD1-/-hPSCs中通过CRISPR基因组编辑获得原代纤毛基因敲除系，以及2。 利用人SSTR3-GFP构建原代纤毛报告系。纤毛敲除线将使机械学研究成为可能 涉及ARPKD肾脏和其他器官中纤毛应激介导的致病信号。记者们排成一行 将能够在不同的实验设置中实时成像初级纤毛运动，包括 移植动物的芯片上有机物和活体成像。 我们提议的工作将为PKD和纤毛疾病研究社区产生新的体外工具 探讨纤毛应激在肾脏和其他脏器中诱发囊变的病理机制。 来自灌流的肾脏器官的转录数据也将提供对膀胱发生的机械性见解。 ARPKD.这项研究开发的所有工具和数据都将通过NIH PKD和RBK传播 促进PKD患者的治疗发展。

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.