Application of progenitor niche signals to ex vivo nephrogenesis

 描述(申请人提供)：大约500,000名美国人患有终末期肾病，其肾功能不足以维持生命。这些人的器官功能可以通过透析得到补充；然而，接受透析的人的10年存活率仅略高于10%。接受肾移植的患者的存活率要好得多，但器官供应与需求不匹配。体外器官发生既可以为肾脏替代治疗提供功能组织，也可以为我们了解慢性肾脏疾病的病因和寻找新的治疗方法提供研究工具。此外，定义从功能上指导肾脏发生的信号可以确定可以被操纵的途径，以增强体内受损肾脏的再生反应。我们的团队能够在纯化的来自人类干细胞和小鼠胚胎肾脏的原代肾单位祖细胞的培养中促进肾脏生成。然而，如果这一发现要对人类健康产生重大影响，我们已经确定了必须克服的两个关键障碍。首先，我们必须重建促进祖细胞增殖和分化的信号环境，以便获得足够的小管质量来进行功能分析。其次，我们必须设计体外组织结构，支持具有适当图案的血管连接的排列肾单位的分化。这项提议的长期目标是定义组织结构和细胞信号微环境，以促进培养中适当模式的肾单位的产生。我们将研究足以促进NPC更新和分化的信号机制，并将这些发现应用于开发使用丝蛋白作为支架生物材料的体外肾单位装置。我们的数据显示，鼻咽癌细胞有效地定植丝绸，并在这种材料中形成肾小管。丝绸是首选材料，因为它已经在临床上使用，而且它是大规模的，使我们能够产生适合老鼠或人类的结构。我们将利用项目研究人员在肾脏和内皮细胞开发以及生物工程方面的优势，在体外开发出集成的、多细胞的、有图案的、有功能的肾单位。目前，创新技术将应用于从人类胚胎干细胞和胚胎小鼠肾脏分化出的神经干细胞。将探索允许组织在培养中有组织的3D器官发生的新的支架和基质配方。调查组有10年的合作历史，这一点在我们初步数据的综合性质上特别明显。我们将利用这一计划，在所有四个实验室之间开展工程和信号专业知识、材料和智力创新的无缝交流。因此，该方案包括一个持续科学交流的框架，包括一个数据跟踪网站、大约每6个月举行一次小组会议和面对面会议的双月时间表。

 DESCRIPTION (provided by applicant): Approximately 500,000 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 both to provide functional tissue for renal replacement therapy and to provide research tools with which we can understand the causes of chronic kidney disease and identify new therapies. 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. Our group is able to promote nephrogenesis in cultures of purified primary nephron progenitor cells derived from human stem cells and mouse embryonic kidneys. However, we have identified two key obstacles that must be overcome if this discovery is to have significant impact on human health. First, we must recreate signaling environments that promote progenitor cell proliferation and differentiation in order to obtain sufficient tubule mass for functional analysis. Second, we must devise ex vivo tissue architecture that supports differentiation of arrayed nephrons with vascular connections that are appropriately patterned. The long-term goal of this proposal is to define the tissue architecture and cell signaling microenvironments required to promote the generation of appropriately patterned nephrons in culture. We will investigate signaling mechanisms that are sufficient to promote NPC renewal and differentiation and apply these findings to the development of ex vivo nephron devices using silk protein as a scaffolding biomaterial. Our data show that NPCs efficiently colonize silk and that nephron tubules form in this material. Silk is the material of choice because it is already in clinical use and it is scalale, allowing us to generate structures dimensioned for mouse or human. We will leverage the strengths of the project investigators in kidney and endothelial development as well as bioengineering to develop an integrated, multi-cellular, patterned, functional nephron in vitro. State of the art, innovative technologies will be applied to NPCs differentiated from both human embryonic stem cells and embryonic mouse kidneys. Novel scaffolding and matrix formulations that allow organized 3D organogenesis of tissues in culture will be explored. The investigative group has a 10-year history of collaboration, which is particularly evident in the integrated natur of our preliminary data. We will leverage this program to develop a seamless exchange of engineering and signaling expertise, materials and intellectual innovation between all four laboratories. The program therefore includes a framework for continual scientific exchange including a data tracking website, a bi-monthly schedule of group meetings and face-to-face meetings approximately every 6 months.