A Human Organoid Model of Polycystic Kidney Disease

多囊肾病的类器官模型 ! 项目摘要 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PKD中， 肾脏和其它器官的正常管状结构逐渐被囊肿所取代， 纤维化PKD无法治愈，候选疗法的疗效和安全性尚不确定。 PKD通常作为PKD 1或PKD 2中的生殖系杂合功能丧失突变遗传， 分别编码多囊蛋白-1（PC 1）和多囊蛋白-2（PC 2）。这些巨大的跨膜 蛋白质在初级纤毛和其它部位形成通道-受体复合物。目前还不知道 突变如何导致肾小管上皮细胞形成囊肿。破译PKD的主要障碍 从机械上讲，缺乏忠实概括PKD的实验可用模型， 由小管产生的特异性囊肿。原代细胞系和永生化细胞系是异质的， 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 基本上来自人类，并且具有挑战性地进行机械破译。为了克服这一差距， 我们的实验室是使用人类多能干细胞（hPSC）来模拟PKD的先驱。 hPSC代表非常早期胚胎状态并提供患者匹配的可再生来源 用于分析和再生的人类细胞。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。我们进一步比较了基因编辑的和患者来源的类器官与小鼠 和患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疾病相关的表型，包括从 肾类器官小管。这个建议的目的是阐明机械的决定因素， 肾类器官中人PKD囊肿形成。根据我们的初步数据，我们假设， PC 1和PC 2的平衡表达调节肾小管与其周围组织之间的物理粘附。 微环境目的一：建立一种更为可靠的人PKD实验模型， 定量杂合肾类器官中的囊肿形成。在目标2中，我们将研究如何 通过阐明PKD 2-/-中PC 1丢失的潜在机制来控制多囊蛋白水平。 细胞最后，Aim 3将通过鉴定PKD的蛋白质， 在包囊形成过程中细胞粘附的缺陷。将在原发性PKD组织中验证关键结果 和非类器官细胞。总的来说，这些研究将揭示关键的分子途径， 囊肿形成的神秘过程，揭示了治疗干预的新的潜在目标。

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.