A Human Organoid Model of Polycystic Kidney Disease

多囊肾病的人体类器官模型

基本信息

批准号：
10447043
负责人：
Benjamin Solomon Freedman
金额：
$ 35.3万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-15 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10447043
关键词：
Actomyosin Adhesions Affect Animal Model Animals Architecture Biochemical Cell Adhesion Cell Line Cell membrane Cell model Cell surface Cells Cilia Complement Complex Cyst Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Cytoskeleton Data Defect Disease Embryo Endoplasmic Reticulum Epithelial Cells Excision Experimental Models Extracellular Matrix Fibrosis Genes Genetic Genetic Diseases Genetic Transcription Goals Hereditary Disease Heterozygote Human Inherited Integral Membrane Protein Intervention Kidney Kidney Diseases Kidney Failure Laboratories Life Liquid substance Lysosomes Membrane Modeling Molecular Mus Mutation Myosin ATPase Myosin Type II Natural regeneration Nature Nephrons Organ Organoids PKD2 protein Pathway interactions Patients Pattern Persons Pharmaceutical Preparations Pharmacology Phenocopy Phenotype Polycystic Kidney Diseases Process Proteins Publishing Receptor Signaling Renal tubule structure Research Role Safety Site Source Structure Surface System Techniques Testing Therapeutic Therapeutic Intervention Time Tissue Sample Tissues Tube Tubular formation Work base cell immortalization cohort experimental study human model human pluripotent stem cell human tissue innovation loss of function mutation mouse model multicatalytic endopeptidase complex mutant non-muscle myosin novel polycystic kidney disease 1 protein prenatal prevent receptor stem cell model stoichiometry success targeted treatment therapeutic candidate trafficking

项目摘要

A HUMAN ORGANOID MODEL OF POLYCYSTIC KIDNEY DISEASE ! PROJECT SUMMARY Polycystic kidney disease (PKD) is the world's most common life-threatening genetic disease and fourth-leading cause of kidney failure, affecting approximately 12 million people worldwide. In PKD, the normal tubular architecture of the kidneys and other organs is gradually replaced by cysts and fibrosis. There is no cure for PKD, and candidate therapeutics are of uncertain efficacy and safety. PKD is commonly inherited as a germline heterozygous loss-of-function mutation in PKD1 or PKD2, encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively. These large, transmembrane proteins form a channel-receptor complex at the primary cilium and other sites. It is not yet known how mutations result in cyst formation from tubular epithelial cells. A major barrier to deciphering PKD mechanistically is the lack of experimentally accessible models that faithfully recapitulate PKD- specific cystogenesis from tubules. Primary and immortalized cell lines are heterogenous, de- differentiated, non-human, or represent only later stages of disease, while animal models differ substantially from humans and are challenging to decipher mechanistically. To overcome this gap, our laboratory is pioneering the use of human pluripotent stem cells (hPSC) for modeling PKD. hPSC represent a very early embryonic state and provide a renewable source of patient-matched human cells for analysis and regeneration. We have established techniques to differentiate hPSC into human kidney organoids, which are complex, multicellular structures with patterned segments that resemble nephrons. We have further compared gene-edited and patient-derived organoids to mouse and human tissue samples with disease to complement and validate this new system. In hPSC with PKD mutations, we have identified several disease-relevant phenotypes, including cystogenesis from kidney organoid tubules. The goal of this proposal is to elucidate the mechanistic determinants of human PKD cystogenesis in kidney organoids. Based on our preliminary data, we hypothesize that balanced expression of PC1 and PC2 regulates physical adhesion between the kidney tubule and its microenvironment. In Aim 1, we will establish a more faithful experimental model of human PKD by quantifying cystogenesis in heterozygous kidney organoids. In Aim 2, we will investigate how polycystin protein levels are controlled by clarifying the mechanisms underlying PC1 loss in PKD2-/- cells. Finally, Aim 3 will explore a novel hypothesized molecular pathway for PKD by identifying defects in cell adhesion during cyst initiation. Key findings will be validated in primary PKD tissues and non-organoid cells. Collectively, these studies will unveil critical molecular pathways that underlie the enigmatic process of cyst formation, revealing new potential targets for therapeutic intervention.

多囊肾病的人体器官模型！项目概要多囊肾病（PKD）是世界上最常见的危及生命的遗传性疾病，肾衰竭的第四大原因，影响全球约 1200 万人。在公钥簿中，肾脏和其他器官的正常管状结构逐渐被囊肿取代纤维化。多囊肾无法治愈，候选疗法的疗效和安全性也不确定。 PKD 通常是作为 PKD1 或 PKD2 种系杂合功能丧失突变而遗传的，分别编码多囊蛋白-1 (PC1) 和多囊蛋白-2 (PC2)。这些大的、跨膜的蛋白质在初级纤毛和其他位点形成通道受体复合物。目前尚不清楚突变如何导致肾小管上皮细胞形成囊肿。破译公钥簿的主要障碍从机制上讲，缺乏可忠实重述 PKD 的实验可访问模型来自肾小管的特异性囊肿发生。原代细胞系和永生化细胞系是异质的、去- 分化的、非人类的或仅代表疾病的后期阶段，而动物模型则不同基本上来自人类，并且很难机械地破译。为了克服这一差距，我们的实验室率先使用人类多能干细胞 (hPSC) 来建模 PKD。 hPSC 代表了非常早期的胚胎状态，并提供了患者匹配的可再生来源用于分析和再生的人体细胞。我们已经建立了将 hPSC 分化为人类肾脏类器官，是复杂的多细胞结构，具有图案化的片段类似于肾单位。我们进一步将基因编辑和患者来源的类器官与小鼠进行了比较以及患有疾病的人体组织样本来补充和验证这个新系统。在 hPSC 中 PKD 突变，我们已经确定了几种与疾病相关的表型，包括来自肾类器官小管。该提案的目标是阐明机械决定因素肾类器官中的人 PKD 囊肿发生。根据我们的初步数据，我们假设 PC1和PC2的平衡表达调节肾小管与其肾小管之间的物理粘附微环境。在目标 1 中，我们将通过以下方式建立更忠实的人类 PKD 实验模型：量化杂合肾类器官的囊肿发生。在目标 2 中，我们将研究如何通过阐明 PKD2-/- 中 PC1 丢失的机制来控制多囊蛋白水平细胞。最后，目标 3 将通过识别 PKD 来探索一种新的假设分子途径囊肿发生过程中细胞粘附缺陷。主要发现将在原发性 PKD 组织中得到验证和非类器官细胞。总的来说，这些研究将揭示潜在的关键分子途径囊肿形成的神秘过程，揭示了治疗干预的新潜在目标。