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.

摘要 肾祖细胞的鉴定有望阐明其对发育缺陷的贡献 以及用于分离人肾祖细胞作为评价其治疗潜力的先决条件。 成人肾脏是否含有祖细胞是一个激烈争论的问题。因为哺乳动物的肾脏 在正常脱落和损伤后再生新细胞，我们已经发表了一个严格的定义， 肾祖细胞需要体内证明1）自我更新，2）克隆形成，3）多能性，和 参与4）组织维护和5）损伤修复。我们已经鉴定了Aqp 2+细胞的一个子集， 用识别V-ATP酶亚基B1和B2的抗体（Aqp 2 + B1 B2+）也呈阳性染色， 第一个严格符合这五个要求的潜在候选人。这些Aqp 2+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5种类型的细胞，包括 主细胞（PC）和嵌入细胞（IC）在发育过程中形成DCT 2、CNT和CD。成人AP 具有这些能力，并在组织维持期间在DCT 2、CNT和CD中再生所有细胞类型 单侧输尿管梗阻（UUO）后。AP表达IC选择性Jag 1和PC选择性Notch 1，并且 介导与Notch激活相关的修复。其他人报道了转录组中明显的性别偏见 PC的轮廓。所有这些发现都为该项目奠定了坚实的基础。在本提案中，我们建议 验证我们核心假设，即AP具有独特的分子特征及其再生潜力 在男性和女性之间存在差异，并由Jag 1调节。具体目标是确定和验证 AP独特的分子特征（Aim 1），以研究AP的再生潜力（Aim 2）和AP的 在组织维持期间和UUO诱导的损伤修复期间由Jag 1（Aim 3）调节。我们将探索一个 尖端技术/方法的组合，包括基于RFP的细胞分选以富集Aqp 2+谱系 细胞，单细胞RNA-Seq，基于Aqp 2 ECE/+的谱系追踪，无偏胸苷类似物标记，以及一组 创新的测试已被证明是有效的积极验证B1 B2作为AP的标志物。 该项目的成功完成可能会1）加强AP作为一个新的概念，它不同于 据报道，近端小管可以为发育、稳态和 再生机制; 2）对AP与PC和IC的差异行为产生更深入的了解; 3）识别 并验证AP的独特分子特征以用于将来的分离; 4）将AP介导的修复与 Notch信号传导; 5）建立性别和Jag 1作为AP的潜在调节因子; 6）回答许多 关于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， 以及这种途径的中断如何导致肾纤维化。总之，这些发现对人类具有重要意义。 病理学和干细胞生物学，以及用于改善体外类器官生成。