Identifying pathways required for integration of kidney organoid and host epithelia

项目摘要 肾脏疾病是美国第九大死亡原因，因为几乎没有治疗方法可以预防或减缓肾脏疾病。 在进展中，超过700，000名患者患有终末期肾病。这些患者接受透析治疗， 肾移植，后者导致明显优越的上级生存。然而，肾脏捐赠者是有限的， 对于增强肾修复或产生用于肾移植的新肾单位的策略， 更换.多能干细胞衍生的类器官显示出分化的肾小管的关键特征， 肾小球结构在体外，我们已经表明，他们产生的图案肾单位在体内显示， 肾功能，如近端小管的过滤和葡萄糖摄取。开发这项技术， 肾替代，干细胞衍生的小管必须连接到宿主小管用于尿输出。我们最近 在斑马鱼中的工作表明，FGF信号传导作为一种趋化信号来招募和激活细胞， 在新的肾单位形成的位点和典型的Wnt信号传导是侵入性细胞重排所必需的， 连接小管腔。包括非经典Wnt信号传导在内的其他信号传导途径也可能 在肾小管相互连接中发挥作用。为了充分探索小管互连的要求，我们 建立了一个协同的三部分发现平台，包括1）体内新肾单位的遗传分析 2）哺乳动物上皮细胞的体外3D细胞培养分析， 融合，和3）体内干细胞衍生的肾类器官移植到宿主小鼠收集系统。我们将 联合收割机来分析小管融合过程的多个步骤，包括：1） 肾单位祖细胞靶向上皮细胞，2）去除介入的ECM/基底膜，3）图案化 靶上皮细胞的集体细胞侵入，和4）建立连续的通畅的新腔以输送靶上皮细胞的细胞侵入。 肾单位滤液。这些研究将提供重要的新的见解，关于一个重要的，但研究不足的细胞 体内植入新肾组织的肾再生所需的机制 治疗

Project Summary Kidney disease is the 9th leading cause of death in the U.S. Because few therapies exist to prevent or slow progression, over 700,000 patients have End Stage Renal Disease. These patients are treated with dialysis or renal transplant, the latter resulting in markedly superior survival. However, kidney donors are limited and there is an important unmet need for strategies that enhance renal repair or generate new nephrons for renal replacement. Pluripotent stem cell derived organoids display key features of differentiated kidney tubules and glomerular structures in vitro, and we have shown that they generate patterned nephrons in vivo displaying kidney functions such as filtration and glucose uptake by the proximal tubule. To develop this technology for renal replacement, stem cell derived tubules must be connected to host tubules for urinary output. Our recent work in the zebrafish demonstrated that FGF signaling acts as a chemotactic signal to recruit and polarize cells at sites of new nephron formation and canonical Wnt signaling is required for invasive cell rearrangement to connect tubule lumens. Additional signaling pathways including non-canonical wnt signaling are also likely to play a role in tubule interconnection. To fully explore the requirements for tubule interconnection we have established a synergistic, three-part discovery platform comprising 1) genetic analysis of in vivo new nephron addition in the regenerating zebrafish adult kidney, 2) in vitro 3D cell culture analysis of mammalian epithelial fusion, and 3) in vivo stem cell-derived kidney organoid engraftment to a host mouse collecting system. We will combine these approaches to analyze multiple steps of the tubule fusion process involving 1) recruitment of nephron progenitor cells to target epithelia, 2) removal of intervening ECM/basement membranes, 3) patterned collective cell invasion of target epithelia, and 4) establishment of a continuous patent new lumen to convey the nephron filtrate. These studies will provide important new insights about an essential but understudied cellular mechanism that will be required for in vivo engraftment of new kidney tissue-based renal regeneration therapies.