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因，因为几乎没有什么治疗方法可以预防或减缓 进展中，超过700,000名患者患有终末期肾病。这些患者接受透析或 肾移植，后者导致明显更好的存活率。然而，肾脏捐赠者是有限的，而且 是加强肾脏修复或为肾脏产生新的肾单位的策略的一个重要的未满足的需求 替补。多能干细胞衍生的有机体显示分化的肾小管和 体外肾小球结构，我们已经证明它们在活体显示产生有图案的肾单位 肾功能，如近端小管的滤过和葡萄糖摄取。为了开发这项技术 肾移植，干细胞来源的肾小管必须连接到宿主肾小管才能排出尿量。我们最近 对斑马鱼的研究表明，成纤维细胞生长因子信号作为一种趋化信号来招募和极化细胞 在新的肾单位形成和典型的Wnt信号是侵袭性细胞重排所必需的 连接小管腔。包括非规范WNT信号在内的其他信号通路也可能 在小管互联中发挥作用。为了充分探讨小管互连的要求，我们有 建立了一个由三部分组成的协同发现平台，包括1)体内新肾单位的遗传分析 在斑马鱼成体肾脏再生中的应用2)哺乳动物肾上皮细胞的体外三维培养分析 融合，以及3)体内干细胞来源的肾脏有机物植入宿主小鼠收集系统。我们会 结合这些方法来分析小管融合过程的多个步骤，涉及1)招募 肾单位前体细胞靶向上皮细胞，2)去除中间的ECM/基底膜，3)图案化 靶上皮细胞的集体侵袭，以及4)建立连续的专利新腔来传递 肾单位滤液。这些研究将为一个基本但未被研究的细胞提供重要的新见解 体内植入基于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.