Defining key differences in mouse fibroblasts during digit regeneration and fibrosis

定义小鼠成纤维细胞在手指再生和纤维化过程中的关键差异

• 批准号: 10602674
• 负责人: Vivian Jou
• 金额: $ 3.72万
• 依托单位: HARVARD UNIVERSITY
• 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 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10602674
• 关键词: Address; Alleles; Amputation; Automobile Driving; Bar Codes; Behavior; Biological Assay; Biological Models; Biological Process; Candidate Disease Gene; Cell Lineage; Cells; Cellular Structures; Chromosome Mapping; Clinical Treatment; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA; Data; Digit structure; Distal; Doxycycline; Electroporation; Fibroblasts; Fibrosis; Gene Delivery; Gene Expression; Genes; Genetic; Genetically Engineered Mouse; Homeostasis; Human; Hydrogels; Knowledge; Mammals; Modeling; Molecular; Mus; Nail plate; Natural regeneration; Persons; Plasmids; Play; Population; Population Dynamics; Population Heterogeneity; Process; Protein Secretion; Research; Resolution; Role; Sepharose; Source; Survival Rate; System; Techniques; Testing; Tissues; blastema; cell type; design; differential expression; digit regeneration; driving force; effective therapy; experimental study; high throughput screening; improved; in vivo; insight; knock-down; limb amputation; overexpression; regenerative; repaired; response; single-cell RNA sequencing; small hairpin RNA; tissue regeneration; tool; transcriptomics; wound; wound healing

PROJECT SUMMARY Composite tissue regeneration is very limited in mammals; however, humans and mice can fully regenerate the distal tips of the digits following amputation. This process involves the formation of a blastema, a cellular structure that is the source of the regenerated tissue and is integral to successful regeneration. Proximal amputations beyond the nail do not form a blastema and result in fibrotic wound-healing. This differential behavior makes the mouse digit tip an ideal model system to investigate the cellular and molecular factors driving each wound-healing response and why complex regeneration is so limited in mammals. Specifically, this project will focus on fibroblast subtypes and their role in fibrosis versus regeneration. Fibroblasts are a major contributor to the blastema and play an integral part in fibrosis; thus, they may be a cell population that drives the decision between fibrosis and regeneration. Our single cell transcriptomic (scRNA-seq) analysis of the regenerating blastema revealed an extremely heterogenous fibroblast population and that the subpopulations had distinct population dynamics and lineage trajectories during blastema formation and maturation. I hypothesize that there are specific fibroblast subtypes that promote regeneration, inhibit fibrosis, or both. However, no studies have performed direct lineage contributions by tracing fibroblast subpopulations in regeneration and fibrosis. Additionally, the in vivo functional roles and importance of our computationally defined candidate pro-regenerative genes have not been established. This project will utilize single-cell CRISPR based DNA barcoding for lineage tracing fibroblasts at the subtype resolution (Aim 1) and plasmid electroporation for gene delivery to functionally assess candidate pro-regenerative genes (Aim 2). Together, my two aims will provide important insight into how the fibrotic and regenerative processes are determined in the mouse digit tip and will open additional avenues for more effective clinical treatments for large wounds or amputations in humans.

项目概要 哺乳动物的复合组织再生非常有限；然而，人类和老鼠可以完全再生 截肢后手指的远端。这个过程涉及胚基的形成，胚基是一种细胞 结构是再生组织的来源，对于成功再生至关重要。近端 指甲以外的截肢不会形成芽基并导致纤维化伤口愈合。这个差速器 行为使小鼠指尖成为研究细胞和分子因素的理想模型系统 驱动每种伤口愈合反应以及为什么复杂的再生在哺乳动物中如此有限。具体来说， 该项目将重点关注成纤维细胞亚型及其在纤维化与再生中的作用。成纤维细胞是一种 胚基的主要贡献者并在纤维化中发挥不可或缺的作用；因此，它们可能是一个细胞群 这推动了纤维化和再生之间的决定。我们的单细胞转录组 (scRNA-seq) 对再生胚基的分析揭示了极其异质的成纤维细胞群，并且 这些亚种群在芽基形成和发育过程中具有不同的种群动态和谱系轨迹 成熟。我假设有特定的成纤维细胞亚型可以促进再生、抑制 纤维化，或两者兼而有之。然而，没有研究通过追踪成纤维细胞来进行直接谱系贡献 再生和纤维化的亚群。此外，我们的体内功能作用和重要性 尚未建立计算定义的候选促再生基因。该项目将利用 基于单细胞 CRISPR 的 DNA 条形码，用于亚型分辨率的谱系追踪成纤维细胞（目标 1） 和质粒电穿孔用于基因传递，以功能评估候选促再生基因（目标 2）。我的两个目标将共同提供关于纤维化和再生过程如何发生的重要见解 在小鼠指尖中确定，将为更有效的临床治疗开辟更多途径 人类的大伤口或截肢。