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 目前有超过10万美国人在等待肾移植，但供不应求。体外器官发生是肾脏替代治疗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(IPSCs)中产生细胞复杂的肾脏有机化合物的技术。从理论上讲，IPSC衍生的肾脏有机化合物将被移植到宿主组织上，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，植入的有机类物质与宿主肾脏的结合很差。基于实验观察，我们确定了至少三个必须克服的关键障碍，以产生完整的肾脏器官类化合物(图1)： 组织结构：器官样小管、血管和间质缺乏皮质-髓质结构。 小管融合：在目前的策略下，器质性小管和血管不与宿主组织吻合。 功能分析：我们缺乏用器官组织成功地进行肾脏替代的分析。 我们合作团队的目标是生成工具、试剂和方案来克服每一个障碍。在我们最近的研究中，我们发现：1)需要不同的基质细胞群来建立正确的小管和血管系统的带状组织和节段性分化；2)有机体缺乏基质细胞群的正态分布和补充性；3)基质细胞及其分泌产物可以促进小管与小管的相互连接。这些发现表明，适合体内功能的肾脏器官类物质的形成取决于创造正确的基质细胞补充和组织。通过对基质细胞群体的操作，可以产生与植入部位匹配的解剖学上“正确的”组织这一概念是新颖的，并确定了我们对发育中的肾脏组织的基质细胞生物学的理解的一个空白。为了填补这一空白，并产生资源来确定如何利用这种新的图案化机制来生成用于肾脏替代的肾组织，我们提出了一系列假说生成研究，这些研究将确定不同的基质细胞在图案化血管系统和肾上皮以及促进肾小管连接到宿主中的作用。我们的长期目标是生成具有适当图案的微血管的功能性近端肾单位，与宿主的尿路和循环系统整合，用于功能性肾脏替代。 为了促进小管和血管融合，我们将开发一条管道，用于在肾功能受损的动物身上对实验室来源的组织进行体内测试。在初步数据中，我们展示了新的功能分析，包括ELISA和肾小管液体流动的实时成像，可以快速和容易地评估植入的有机物质对宿主尿液产生的贡献。我们还将创造新的资源，包括基质细胞系、报告小鼠、成像技术和量化器官组织替代肾脏的程度的分析。我们提出以下目标： 目的1：开发调节器质间质以促进近端肾单位节段的策略。制定促进近端小管间质形成的方案。 1B.探讨巨噬细胞在类器官分化中的作用。 目的2.在组织移植物2a中生成健壮的、功能化的肾血管系统。评估血管在有机体中的维持、生长和招募。 2B。描述间质对器官血管系统的影响。 2C。产生“维管启动的”有机物质。 目的3.确定促进肾上皮细胞小管融合的策略 3A。确定间质相互作用在上皮小管吻合中的作用。 3B。确定肝细胞生长因子和转化生长因子在器质性肾小管吻合中的作用。 目的4.评价移植组织在体内的功能 4A.开发一条管道来测试移植组织的功能替代潜力。 4B。评估再生肾与纤维化肾中有机类物质的植入。 这些目标的完成有望极大地提高人们对基质细胞群体如何为器官组织、血管形成和连接/植入提供所需线索的认识。成功的完成将揭示总体概念并产生急需的资源，这些资源将推动这一领域的研究，并为为广大晚期肾脏疾病患者提供功能性肾脏替代组织的最终目标做出重大贡献，我们相信这一目标在未来10-20年内是可以实现的。 1