Application of Progenitor Niche Signals to Ex Vivo Nephrogenesis

摘要 大约75万美国人患有终末期肾病，在这种疾病中，肾脏 功能不足以维持生命。这些患者的器官功能可以通过透析得到补充 然而，接受透析的个人的10年存活率仅略高于10%。 接受肾移植的患者的存活率要高得多，但器官供应却是如此 与需求不符。体外器官发生有可能为 肾脏替代疗法。此外，定义在功能上指导肾脏发生的信号 可以识别可以被操纵以增强细胞再生反应的途径 活体肾脏损伤。包括我们自己在内的几个组织已经建立了一些技术， 使我们能够产生来自人类和小鼠的细胞复杂的肾脏有机化合物 诱导的多能细胞。这些组织似乎是产生肾脏替代的理想来源。 组织。从理论上讲，人们可以将患者提取的肾脏器官移植到 病变的肾脏，在那里它们将与宿主的泌尿系统结合并改善肾脏 功能。尽管有几个组织已经尝试进行这些类型的移植， 到目前为止，还没有证据表明它们在功能上与宿主肾脏整合。在我们的 在初步研究中，我们确定了必须克服的三个关键障碍，以便 产生与宿主结合的体外肾脏器官类物质。首先，类有机物的结构是 相对杂乱无章，这与沿 健康的天然肾脏的皮质-髓质轴。其次，在目前的策略下，有机化合物- 衍生的小管不与宿主衍生的小管和有机衍生的小管相连 随着时间的推移逐渐变得复杂。第三，我们缺乏可靠的功能分析来确定实验性的修改 改善器官功能的药物。必须消除这些障碍中的每一个才能产生 可在临床上对患者有益的有机化合物。 我们假设，获得完整的有机组织的最好方法是 选择性地产生与植入的解剖部位相匹配的细胞类型。具体来说，我们将 确定允许我们产生近端肾单位的条件，包括肾小球和 近端小管及其相关的间质和血管系统(此处称为皮质 有机化合物)用于嫁接。为此，我们的体外肾单位策略 GENERATE的独特之处在于它强调促进解剖学上“正确”的上皮和它的 嫁接部位的微环境。与此同时，我们将确定因素和 促进小管-小管融合的技术。因此，一旦我们产生了皮质有机体， 我们将利用这项技术来刺激移植物的小管与小管吻合。 主人的名字。最后，我们将使用实时成像和定义明确的功能分析作为读数 管状功能，不断优化我们的战略。这项提议的长期目标是 工程师组织的体外肾组织，可被诱导形成功能性肾单位 通过新的嫁接策略在动物宿主中。

Summary Approximately 750,000 Americans have end stage renal disease, in which kidney function is insufficient to sustain life. Organ function can be supplemented by dialysis in these individuals, however the 10 year survival rate for individuals on dialysis is just over 10%. Survival rates are much better for patients receiving a kidney transplant, but organ supply does not match demand. Ex vivo organogenesis has the potential to provide functional tissue for renal replacement therapy. Furthermore, defining signals that functionally direct nephrogenesis may identify pathways that can be manipulated to augment the regenerative response of the injured kidney in vivo. Several groups, including our own, have established techniques that allow us to generate cellularly complex kidney organoids derived from human and mouse induced pluripotent cells. These tissues seem an ideal source for generating renal replacement tissue. Theoretically, one would take patient-derived renal organoids and transplant them onto a diseased kidney, where they would integrate with the host urinary system and improve renal function. Although several groups have attempted to perform these types of transplantations, there is no evidence to date that they functionally integrate with the host kidney. In our preliminary studies, we have identified three key obstacles that must be overcome in order to generate ex vivo renal organoids that integrate with the host. First, organoid structure is relatively disorganized, which is in contrast to the precise arrangement of cell types along the cortical-medullary axis of healthy, native kidneys. Second, with current strategies, organoid- derived tubules do not connect with host-derived tubules and the organoid-derived tubules involute over time. Third, we lack robust functional assays to identify experimental modifications that improve organoid function. Each of these barriers must be eliminated to generate functional organoids that can be clinically beneficial to patients. We hypothesize that the best approach to achieve integrated organoid tissue is to selectively generate cell types that match the anatomic site of engraftment. Specifically, we will identify conditions that will allow us to generate proximal nephrons, including glomeruli and proximal tubules with their associated interstitium and vasculature, (herein referred to as cortical organoids) for the purposes of engraftment. To this end, our strategy for ex vivo nephron generation is unique in its emphasis on promoting the anatomically “correct” epithelia and its microenvironment for the site of engraftment. Concurrent to this, we will identify factors and techniques that promote tubule-tubule fusion. Thus, once we have generated cortical organoids, we will utilize this technology to stimulate the tubules of the graft to anastomose with the tubules of the host. Finally, we will use live imaging and well-defined functional assays as a readout of tubular function to continually optimize our strategy. The long-term goal of this proposal is to engineer organized, ex vivo renal tissue that can be induced to form functional nephrons in animal hosts through novel grafting strategies.