The Regenerative Potential of Aqp2+ Progenitor Cells

Aqp2 祖细胞的再生潜力

• 批准号: 10716327
• 负责人: WENZHENG ZHANG
• 金额: $ 50.93万
• 依托单位: ALBANY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10716327
• 关键词: Adult; Antibodies; Behavior; Cell Count; Cell Lineage; Cell Separation; Cells; Classification; Clone Cells; Data; Data Set; Defect; Development; Disease; Distal convoluted renal tubule structure; Duct (organ) structure; Embryo; Exhibits; Female; Foundations; Future; Generations; Histologic; Human; Human Pathology; Immune; Immunofluorescence Immunologic; In Situ Hybridization; In Vitro; Inherited; Injury; Intercalated Cell; Kidney; Label; Ligands; Link; Maintenance; Mediating; Molecular Profiling; Mus; Natural regeneration; Organoids; Osmolalities; Pathway interactions; Property; Publishing; Regenerative Medicine; Regulation; Reporting; Research; Sex Bias; Signal Transduction; Solid; Stains; Tamoxifen; Techniques; Testing; Therapeutic; Thymidine; Tissues; Transitional Cell; Ureteral obstruction; Urinary tract infection; Work; analog; biomarker identification; cell type; daughter cell; extracellular; improved; in vivo; injury and repair; innovation; insight; kidney cell; kidney fibrosis; male; notch protein; novel; novel marker; regeneration potential; regenerative; repaired; self-renewal; sex; single-cell RNA sequencing; stem cell biology; stem cells; transcriptome; transcriptome sequencing; vacuolar H+-ATPase; water channel

Abstract Identification of renal progenitor cells holds promise for elucidating their contribution to developmental defects and for isolating human renal progenitor cells as a prerequisite to evaluating their therapeutic potential. Whether an adult kidney harbors progenitor cells is a hotly debated issue. Because mammalian kidneys can regenerate new cells following normal shedding and injury, we have published a strict definition of an adult renal progenitor cell requiring in vivo demonstration of 1) self-renewal, 2) clonogenicity, 3) multipotency, and participation in 4) tissue maintenance and in 5) injury repair. We have identified a subset of Aqp2+ cells that were also positively stained with an antibody recognizing both V-ATPase subunits B1 and B2 (Aqp2+B1B2+) as the first potential candidate that strictly meets these 5 requirements. These Aqp2+ progenitor cells (AP) exhibited the capacity of self-renewal, clonogenicity, and multipotency, and generated 5 types of cells including principal cells (PC) and intercalated cells (IC) to form DCT2, CNT, and CD during development. Adult AP also possessed these capabilities and regenerated all cell types in DCT2, CNT, and CD during tissue maintenance and after unilateral ureteral obstruction (UUO). AP express IC-selective Jag1 and PC-selective Notch1, and mediate repair correlating with Notch activation. Others have reported marked sex bias in the transcriptome profile of PC. All of these findings have laid a solid foundation for this project. In this proposal, we propose to test our central hypothesis that AP possess a unique molecular signature and their regenerative potential differs between males and females and is regulated by Jag1. The specific Aims are to identify and validate the AP's unique molecular signature (Aim 1), to investigate the AP's regenerative potential (Aim 2) and AP's regulation by Jag1 (Aim 3) during tissue maintenance and during UUO-induced injury repair. We will explore a combination of cutting edge techniques/approaches including RFP-based cell sorting to enrich Aqp2+ lineage cells, single cell RNA-Seq, Aqp2ECE/+-based lineage tracing, unbiased thymidine analog labeling, and a set of innovative tests that have been proven to be effective for vigorously validating B1B2 as a marker of AP. Successful completion of the project will likely 1) reinforce AP as a novel concept, which differs from what has been reported for the proximal tubules and could shed new light into the developmental, homeostatic, and regenerative mechanisms; 2) yield deeper insights into the differential behavior of AP vs. PC and IC; 3) identify and validate a unique molecular signature of AP for their isolation in the future; 4) link AP-mediated repair to Notch signaling; 5) establish both sex and Jag1 as potential regulators of AP; and 6) answer many fundamental questions regarding the origins of PC and IC, how these cells respond to injury through Notch, and how disruption of this pathway leads to kidney fibrosis. In short, the findings are significant for human pathology and stem cell biology in general, and for improvement of in vitro organoid generation.

抽象的 肾祖细胞的鉴定有望阐明其对发育缺陷的影响 以及分离人肾祖细胞作为评估其治疗潜力的先决条件。 成年肾脏是否含有祖细胞是一个备受争议的问题。因为哺乳动物的肾脏可以 在正常脱落和损伤后再生新细胞，我们发布了成人的严格定义 肾祖细胞需要体内证明 1) 自我更新，2) 克隆形成，3) 多能性，以及 参与 4) 组织维护和 5) 损伤修复。我们已经鉴定出 Aqp2+ 细胞的一个子集 也被识别 V-ATPase 亚基 B1 和 B2 (Aqp2+B1B2+) 的抗体呈阳性染色，如下所示 第一个严格满足这 5 项要求的潜在候选人。这些 Aqp2+ 祖细胞 (AP) 表现出自我更新、克隆形成和多能性的能力，并产生了 5 种类型的细胞，包括 主细胞 (PC) 和闰细胞 (IC) 在发育过程中形成 DCT2、CNT 和 CD。成人 AP 也 拥有这些能力，并在组织维护期间再生 DCT2、CNT 和 CD 中的所有细胞类型 以及单侧输尿管梗阻（UUO）后。 AP 表达 IC 选择性 Jag1 和 PC 选择性 Notch1，以及 介导与Notch激活相关的修复。其他人报告了转录组中明显的性别偏见 个人电脑的配置文件。所有这些发现都为本项目奠定了坚实的基础。在本提案中，我们建议 测试我们的中心假设，即 AP 拥有独特的分子特征及其再生潜力 男性和女性之间存在差异，并受 Jag1 调节。具体目标是识别和验证 AP 独特的分子特征（目标 1），研究 AP 的再生潜力（目标 2）和 AP 的再生潜力（目标 2） Jag1（目标 3）在组织维护和 UUO 诱导的损伤修复过程中的调节。我们将探索一个 结合尖端技术/方法，包括基于 RFP 的细胞分选来丰富 Aqp2+ 谱系 细胞、单细胞 RNA-Seq、基于 Aqp2ECE/+ 的谱系追踪、无偏胸苷类似物标记以及一组 已被证明可有效有效验证 B1B2 作为 AP 标志物的创新测试。 该项目的成功完成可能会 1) 强化 AP 作为一个新颖的概念，它不同于现有的概念 已报道近端小管，可能为发育、稳态和 再生机制； 2) 更深入地了解 AP 与 PC 和 IC 的差异行为； 3）识别 并验证 AP 的独特分子特征，以便将来进行分离； 4）将AP介导的修复链接到 缺口信号； 5) 将性别和 Jag1 确定为 AP 的潜在调节因子； 6）回答很多 有关 PC 和 IC 起源的基本问题，这些细胞如何通过 Notch 对损伤做出反应， 以及该途径的破坏如何导致肾脏纤维化。简而言之，这些发现对人类具有重要意义 一般病理学和干细胞生物学，以及改善体外类器官生成。