Application of Progenitor Niche Signals to Ex Vivo Nephrogenesis

祖细胞生态位信号在离体肾发生中的应用

• 批准号: 10295980
• 负责人: Thomas Joseph Carroll
• 金额: $ 152.35万
• 依托单位: ROGOSIN INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-15 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10295980
• 关键词: American; Anastomosis - action; Anatomy; Animals; Biological Assay; Blood Vessels; Cardiovascular system; Cell Line; Cells; Cellular biology; Chronic Kidney Failure; Complement; Complex; Cues; Data; Embryo; End stage renal failure; Engraftment; Enzyme-Linked Immunosorbent Assay; Epithelial; Generations; Goals; Growth; Human; Image; Imaging technology; Impairment; Implant; Investigation; Kidney; Kidney Diseases; Kidney Transplantation; Knowledge; Laboratories; Left; Life; Maintenance; Mus; Nephrons; Normal Statistical Distribution; Organogenesis; Organoids; Patients; Pattern; Physiological; Population; Production; Protocols documentation; Reagent; Renal Replacement Therapy; Renal Tissue; Renal function; Reporter; Research; Resources; Role; Series; Signal Pathway; Signal Transduction; Site; Source; Stromal Cells; Techniques; Technology; Testing; Tissue Grafts; Tissues; Transforming Growth Factor beta; Tubular formation; Urinary system; Urine; Vascular System; Vascularization; base; fluid flow; graft function; in vivo; in vivo evaluation; macrophage; nephrogenesis; novel; novel therapeutics; progenitor; recruit; regenerative; renal epithelium; replacement tissue; tool

SPECIFIC AIMS There are more than 100k American waiting for a kidney transplant, but demand far exceeds supply. Ex vivo organogenesis is a potential source for functional tissue for renal replacement therapy. We and others established techniques to generate cellularly complex kidney organoids from human induced pluripotent cells (iPSCs). Theoretically, iPSC-derived renal organoids would be grafted to host tissue and would integrate with vascular and urinary systems to supplement lost kidney function. However, data indicates that implanted organoids integrate poorly with the host kidney. Based on experimental observations, we identified at least 3 key obstacles that must be overcome to generate integrated renal organoids (Fig. 1): Tissue organization: Organoid tubules, blood vessels, and stroma lack cortico-medullary pattern. Tubular fusion: With current strategies, organoid tubules and vessels do not anastomose with host tissues. Functional assays: We lack assays to identify successful renal replacement by organoid tissue. The goal of our collaborative team is to generate the tools, reagents and protocols to overcome each of these obstacles. In our recent studies, we discovered the following: 1) Distinct stromal cell populations are required to establish correct zonal organization and segment-specific differentiation of tubules and vasculature; 2) Organoids lack a normal distribution and complement of stromal cell populations; 3) Stromal cells and their secreted products can facilitate tubule-tubule interconnection. These findings show that formation of renal organoids that are appropriately patterned for in vivo function depends on creating the correct complement and organization of stromal cells. The concept that anatomically “correct” tissue that is matched to the engraftment site can be generated through manipulation of stromal cell populations is novel and identifies a gap in our understanding of stromal cell biology of developing kidney tissue. To fill this gap and generate resources to determine how this novel patterning mechanism can be exploited to generate kidney tissue for renal replacement, we propose a series of hypothesis-generating investigations that will define the roles of diverse stromal cells in patterning the vasculature and renal epithelium as well as promoting tubular connection to the host. Our longterm goal is to generate functional proximal nephrons with appropriately patterned microvasculature that integrate with the host urinary and circulatory systems for functional renal replacement. To facilitate our efforts to promote tubule and vascular fusion, we will develop a pipeline for in vivo testing of laboratory-derived tissue in animals with impaired kidney function. In preliminary data, we demonstrate novel functional assays including ELISAs and live imaging of tubular fluid flow that can rapidly and easily assess the contribution of implanted organoids to host urine production. We will also generate novel resources, including stromal cell lines, reporter mice, imaging technology, and assays to quantify the extent of renal replacement by organoid tissue. We propose the following Aims: Aim 1: Develop strategies to modulate organoid stroma to promote proximal nephron segments 1A. Develop protocols to promote formation of proximal tubule stroma. 1B. Investigate the role of macrophages in organoid differentiation. Aim 2. Generate robust and functionally regionalized renal vasculature in tissue grafts 2A. Evaluate vascular maintenance, growth, and recruitment in organoids. 2B. Characterize stromal impact on organoid vasculature. 2C. Generate “vascular-primed” organoids. Aim 3. Identify strategies to promote tubule fusion in renal epithelia 3A. Identify the role of stromal interactions on epithelial tubule anastomosis. 3B. Determine the role of HGF and TGFß on tubule anastomosis in organoids. Aim 4. Evaluate function of graft tissue in vivo 4A. Develop a pipeline to test functional replacement potential of graft tissue. 4B. Assess organoid engraftment in regenerative versus fibrotic kidneys. The completion of these aims is expected to greatly advance knowledge of how stromal cell populations provide needed cues for organoid organization, vascularization and connection/engraftment. Successful completion will reveal general concepts and generate much needed resources that will propel research in this field and contribute significantly to the ultimate goal of providing functional renal replacement tissue to the vast number of patients with advanced kidney disease, a goal we believe is achievable in the next 10-20 years. 1

具体目标 有超过1万美国的美国等待肾脏移植，但需求远远超过了供应。离体器官发生是肾脏替代疗法功能组织的潜在来源。我们和其他人建立了从人类诱导的多能细胞（IPSC）产生细胞复杂的肾脏器官的技术。理论上的IPSC衍生的肾脏器官将被嫁接到宿主组织，并与血管和尿液系统整合以补充肾脏丧失的功能。但是，数据表明，植入的类器官与宿主肾脏的整合很差。基于实验观察，我们发现了至少3个关键障碍，这些障碍必须克服以产生综合的肾脏器官（图1）： 组织组织：类器官管，血管和基质缺乏皮质中的软管模式。 管状融合：使用当前的策略，器官管和血管不会与宿主组织吻合。 功能测定：我们缺乏确定器官组织成功肾脏替代的测定法。 我们协作团队的目标是生成工具，试剂和协议，以克服这些障碍。在最近的研究中，我们发现了以下内容：1）需要不同的基质细胞群来建立正确的区域组织和管子特定的管道和脉管系统的分化； 2）类器官缺乏正态分布和基质细胞群体的完成； 3）基质细胞及其分泌的产品可以促进管道互连。这些发现表明，适当为体内功能的适当图案的肾脏器官的形成取决于创建正确的基质细胞的完整和组织。可以通过操纵基质细胞种群来产生与植入部位相匹配的解剖学“正确”组织的概念是新颖的，并且在我们对发展肾脏组织的基质细胞生物学的理解方面存在差距。为了填补这一空白并产生资源，以确定如何探索这种新颖的模式机制以生成肾脏组织以进行肾脏替代，我们提出了一系列假设生成的投资，这些投资将定义潜水大细胞在使脉管系统和肾上皮的作用以及促进与宿主的肾小管连接方面的作用。我们的长期目标是用适当的图案化微脉管系统生成功能性近端肾单位，与宿主尿液和电路系统集成以进行功能性肾脏替代。 为了促进我们促进管和血管融合的努力，我们将开发一条管道，用于在肾功能受损的动物中对实验室衍生的组织进行体内测试。在初步数据中，我们展示了新的功能评估，包括ELISA和结核流体流的实时成像，这些功能可以快速，轻松地评估植入的类器官对宿主尿液产生的贡献。我们还将生成新的资源，包括基质细胞系，报告基因小鼠，成像技术和主张，以量化器官组织肾脏替代的程度。我们提出以下目标： 目标1：制定调节器官基质以促进近端肾单位段1a的策略。开发方案以促进近端小管基质的形成。 1B。研究巨噬细胞在器官分化中的作用。 AIM 2。在组织移植物2a中产生强大的和功能区域化的肾脏脉管系统。评估器官中的血管维持，生长和募集。 2b。表征基质对器官脉管系统的影响。 2C。产生“血管染色”类器官。 目标3。确定促进肾上皮中小管融合的策略 3a。确定基质相互作用对上皮管吻合的作用。 3b。确定HGF和TGFß在器官中块茎吻合术中的作用。 目标4。评估体内移植组织的功能 4a。开发管道以测试移植组织的功能替代潜力。 4b。评估再生与纤维化肾脏中的器官植入。 这些目标的完成有望充分了解基质细胞种群如何为器官组织，血管化和连接/植入提供所需的线索。成功的完成将揭示一般概念并产生急需的资源，这些资源将推动该领域的研究，并为将功能性肾脏替代组织提供给大量患有晚期肾脏病的患者的最终目标做出了巨大贡献，我们认为这一目标是在未来10 - 20年内实现的目标。 1