The Regenerative Potential of Aqp2+ Progenitor Cells

摘要 肾祖细胞的鉴定有望阐明它们在发育缺陷中的作用 并用于分离人肾前体细胞，作为评估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可以 在正常的脱落和损伤后再生新的细胞，我们已经发布了对成人的严格定义 需要在体内证明1)自我更新、2)克隆生成、3)多潜能的肾祖细胞 参与组织维护和损伤修复。我们已经确定了Aqp2细胞的一个子集 也被识别V-ATPase亚基B1和B2(Aqp2 B1B2)的抗体染色为阳性 第一位严格满足这5项要求的潜在候选人。这些Aqp2祖细胞(AP) 表现出自我更新、克隆形成和多潜能的能力，并产生了5种类型的细胞，包括 在发育过程中，主细胞(PC)和嵌入细胞(IC)形成DCT2、CNT和CD。成人美联社也 拥有这些能力，并在组织维护期间再生了DCT2、CNT和CD中的所有细胞类型 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 与Notch激活相关的中介修复。其他人则报告了转录组中明显的性别偏见。 PC的配置文件。所有这些发现都为该项目奠定了坚实的基础。在这项建议中，我们建议 测试我们的中心假设，即AP具有独特的分子签名及其再生潜力 雄性和雌性之间存在差异，受Jag1调节。具体目标是确定和验证 AP独特的分子特征(目标1)，以研究AP的再生潜力(目标2)和AP的 Jag1(Aim 3)在组织维持和UUO诱导的损伤修复过程中的调节。我们将探索一种 结合尖端技术/方法，包括基于RFP的细胞分选，丰富Aqp2谱系 细胞，单细胞RNA-Seq，基于Aqp2ECA/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，有力地验证了B1B2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，这是不同于 已经报道了近端小管的情况，并可能为发育、内稳态和 再生机制；2)深入了解AP与PC和IC的不同行为；3)识别 并验证AP的独特分子特征，为将来分离AP奠定基础；4)将AP介导的修复链接到 Notch信号；5)建立性别和Jag1作为AP的潜在调节者；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， 以及这一途径的中断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 一般的病理学和干细胞生物学，以及改善体外有机体生成。

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.