A Human Organoid Model of Polycystic Kidney Disease

一种多囊肾病人体器官模型的建立 好了！ 项目总结 多囊肾病(PKD)是世界上最常见的威胁生命的遗传病和 是导致肾衰竭的第四大原因，全世界约有1200万人受到影响。在PKD中， 肾脏和其他器官的正常管状结构逐渐被囊肿和 纤维化症。PKD没有治愈的方法，候选疗法的有效性和安全性也不确定。 PKD通常作为一种胚系杂合性功能丧失突变在PKD1或PKD2中遗传， 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部位形成通道-受体复合体。目前还不知道 突变如何导致肾小管上皮细胞形成包囊。破译PKD的主要障碍 从机理上讲，缺乏可通过实验获得的模型来忠实地概括PKD- 从小管到特殊的囊性发生。原代和永生化细胞系是异种的，去 分化的，非人类的，或仅代表疾病的后期阶段，而动物模型不同 很大程度上来自人类，并面临着机械破译的挑战。为了克服这一差距， 我们实验室正在率先使用人类多能干细胞(HPSC)来建立PKD模型。 HPSC代表了非常早期的胚胎状态，并提供了与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区分为 人类肾脏有机体，是复杂的多细胞结构，带有图案片段， 类似于肾单位。我们进一步将基因编辑和患者来源的有机化合物与小鼠进行了比较 和有疾病的人体组织样本来补充和验证这一新系统。在hPSC中使用 PKD突变，我们已经确定了几种与疾病相关的表型，包括来自 肾脏的器质小管。这项提议的目的是阐明 人PKD在肾脏器官中的成囊作用。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肾小管之间的物理粘连 微环境。在目标1中，我们将建立一个更真实的人PKD实验模型。 对杂合子肾脏器官中的囊变进行量化。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的水平 细胞。最后，目标3将探索一种新的假想的PKD的分子途径 包囊形成过程中的细胞黏附缺陷。关键发现将在初级PKD组织中得到验证 和非细胞器细胞。总的来说，这些研究将揭示构成其基础的关键分子途径 包囊形成的神秘过程，揭示了治疗干预的新的潜在靶点。

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.