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 10 万美国人正在等待肾移植，但离体器官生成是肾脏替代疗法的功能组织的潜在来源，我们和其他人建立了从人类诱导多能细胞生成细胞复杂的肾脏类器官的技术。理论上，iPSC 衍生的肾类器官将被移植到宿主组织中，并与血管和泌尿系统整合，以补充丧失的肾功能。然而，数据表明植入的类器官会整合。根据实验观察，我们确定了生成整合性肾类器官必须克服的至少 3 个关键障碍（图 1）： 组织组织：类器官小管、血管和基质缺乏皮质髓质模式。 肾小管融合：按照目前的策略，类器官肾小管和血管不与宿主组织吻合。 功能测定：我们缺乏鉴定类器官组织成功肾替代的测定。 我们合作团队的目标是开发工具、试剂和方案来克服这些障碍。在我们最近的研究中，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片段特异性分化。肾小管和脉管系统的形成；2) 类器官缺乏基质细胞群的正常分布和补充；3) 基质细胞及其分泌产物可以促进肾小管-肾小管互连的形成。体内功能适当模式化的类器官取决于基质细胞的正确补充和组织。通过操纵基质细胞群可以生成与植入位点匹配的解剖学“正确”组织的概念是新颖的，并且确定了一种新的方法。我们对发育中肾组织的基质细胞生物学的理解存在空白，为了填补这一空白并产生资源来确定如何利用这种新颖的模式机制来生成用于肾脏替代的肾组织，我们提出了一系列假设生成研究，这些研究将定义如何利用这种新颖的模式机制来生成肾组织。这我们的长期目标是生成具有适当模式的微脉管系统的功能性近端肾单位，与宿主泌尿和循环系统整合以实现肾功能替代。 为了促进我们促进肾小管和血管融合的努力，我们将开发一个管道，用于对肾功能受损的动物的实验室来源的组织进行体内测试。在初步数据中，我们展示了新颖的功能测定，包括 ELISA 和肾小管流体流动的实时成像。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，我们还将产生新的资源，包括基质细胞系、报告小鼠、成像技术和量化类器官组织肾脏替代程度的测定。下列的目标： 目标 1：制定调节类器官基质以促进近端肾单位段 1A 的策略。制定促进近端肾小管基质形成的方案。 1B. 研究巨噬细胞在类器官分化中的作用。 目标 2. 在组织移植物中生成强大且功能区域化的肾脉管系统 2A。 评估类器官中的血管维持、生长和募集。 2B. 表征基质对类器官脉管系统的影响。 2C. 生成“血管引发”的类器官。 目标 3. 确定促进肾上皮细胞小管融合的策略 3A.确定基质相互作用对上皮小管吻合的作用。 3B. 确定 HGF 和 TGFβ 对类器官中肾小管吻合的作用。 目标 4. 评估移植组织的体内功能 4A.开发测试移植组织功能替代潜力的管道。 4B. 评估再生肾脏与纤维化肾脏中的类器官移植。 这些目标的完成预计将极大地增进对基质细胞群如何为类器官组织、血管化和连接/植入提供所需线索的了解，成功完成将揭示一般概念并产生急需的资源，从而推动该领域的研究并做出重大贡献。为广大晚期肾病患者提供功能性肾脏替代组织的最终目标，我们相信这一目标在未来 10-20 年内可以实现。 1