Live imaging of stem cell dynamics in cornea regeneration

角膜再生中干细胞动态的实时成像

• 批准号: 10217142
• 负责人: Panteleimon Rompolas
• 金额: $ 39.41万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10217142
• 关键词: Ablation; Address; Adult; Affect; Alleles; Axotomy; Biological; Biological Assay; Biology; Calcium Channel; Cell Differentiation process; Cell Lineage; Cell Proliferation; Cells; Cornea; Corneal Diseases; Corneal Injury; Data; Diphtheria Toxin; Disease; Elements; Environment; Environmental Risk Factor; Epidermis; Epithelial; Epithelial Cells; Eye; Eye diseases; Family; Functional disorder; Genes; Genetic; Genetic Models; Goals; Heterogeneity; Histologic; Homeostasis; Image; Imaging technology; Impairment; In Vitro; Injury; Keratitis; Keratoplasty; Knockout Mice; Knowledge; Lasers; Lead; Limbus Corneae; Link; Location; Maintenance; Mediating; Mission; Modeling; Molecular; Mus; National Eye Institute; Natural regeneration; Nerve; Nerve Fibers; Nerve Plexus; Nerve Regeneration; Neuropathy; Neurotrophic Keratitis; Organ; Pathogenesis; Pathology; Patients; Pattern; Peripheral Nerves; Pharmacology; Piezo 1 ion channel; Population; Population Heterogeneity; Process; Proliferating; Regulation; Replacement Therapy; Reporter; Research; Resolution; Role; Science; Signal Pathway; Signal Transduction; Skin; Stratified Epithelium; Surface; System; Testing; Therapeutic; Therapeutic Intervention; Time; Tissues; Trigeminal nerve structure; Vision; Visual impairment; Visually Impaired Persons; World Health Organization; afferent nerve; alternative treatment; axon regeneration; base; conjunctiva; corneal epithelium; corneal regeneration; corneal repair; design; effective therapy; exhaustion; experimental study; genetic approach; genomic tools; imaging approach; imaging genetics; imaging system; improved; in vivo; in vivo regeneration; injury and repair; innovation; limbal; migration; neuron loss; notch protein; novel; novel strategies; novel therapeutics; optogenetics; regional difference; repaired; stem cell population; stem cells; tool; two photon microscopy; wound healing

ABSTRACT According to the World Health Organization 70 million people are visually impaired worldwide due to corneal related diseases and injuries. In the U.S. alone more than 50,000 corneal transplants are performed annually due to lack of alternative treatments. The cornea relies on resident stem cells to sustain vision and efficiently regenerate after injury. Conditions that cause stem cells to deviate from their normal activity lead to corneal disease. Elucidating the identity and mechanism of regulation of corneal stem cells is critical for devising new effective treatments for corneal pathologies. We currently have incomplete knowledge of the stem cell dynamics and regulation in the live cornea. A major roadblock is the inability to visualize directly single cell activity during corneal regeneration to elucidate the precise contribution of stem cells. To overcome this, we have pioneered a novel approach to visualize and track stem cell activity in the intact cornea of live mice by 2- photon microscopy. The overall goal of this project is to implement an integrative approach by combining our live imaging system with state-of-the-art optogenetic and genomic tools to 1) characterize stem cell dynamics, 2) identify intrinsic and extrinsic regulators of stem cell activity and 3) test their requirements for corneal regeneration. In live imaging experiments of the cornea we found that stem and progenitor cells in the self- contained corneal epithelium proliferate and differentiate in topologically diverse patterns during homeostasis and wound healing. We hypothesize that the cornea consists of a heterogeneous population of stem cells with distinct contributions to homeostatic maintenance and injury repair. Aim I of this proposal is designed to resolve the specific contributions of stem cells and their immediate progeny to corneal regeneration and to test their necessity for wound healing. Aim I will also address the role of key molecular signals in regulating the fate of distinct epithelial populations within the cornea. Such signals can potentially be exploited for therapeutic purposes. Aim II will investigate extrinsic regulation of corneal stem cells. The subbasal nerve plexus is a major component of the corneal tissue environment. Our live imaging approach is ideal for studying the functional interactions between the corneal nerves and the epithelium. We have devised in vivo assays for visualizing and manipulating nerve processes in the live cornea. Aim II will test the requirement of the subbasal nerve plexus for epithelial function in homeostasis and wound healing and explore novel mechanisms for corneal nerve regeneration. These data will enable new therapies for patients with Neuropathic Keratitis. This research is innovative because it uses cutting-edge imaging technologies and genetic tools to study stem cells within the natural tissue environment of the live mammalian eye. Our goal is to uncover the fundamental mechanisms of corneal regeneration that will unlock the full potential of stem cells for treating corneal disease.

抽象的 根据世界卫生组织的数据 相关疾病和伤害。仅在美国，每年进行50,000多次角膜移植 由于缺乏替代治疗。角膜依靠常驻干细胞来维持视力并有效 受伤后再生。导致干细胞偏离其正常活性的疾病导致角膜 疾病。阐明角膜干细胞调节的身份和机制对于设计新的身份和机制至关重要 角膜病理的有效治疗方法。我们目前对干细胞有不完整的了解 活角膜中的动力和调节。一个主要的障碍是无法直接可视化单个单元格 角膜再生过程中的活性以阐明干细胞的精确贡献。为了克服这一点，我们 是否开创了一种新颖的方法，可以通过2--可视化和跟踪活小鼠完整角膜中的干细胞活性 光子显微镜。该项目的总体目标是通过结合我们的 具有最先进的光遗传学和基因组工具的实时成像系统至1）表征干细胞动力学， 2）确定干细胞活性的内在和外在调节剂，3）测试其角膜的需求 再生。在角膜的实时成像实验中，我们发现自我的茎和祖细胞 在体内平衡期间，包含角膜上皮增殖和拓扑多样的模式分化 和伤口愈合。我们假设角膜由干细胞的异质种群与 对体内稳态维护和伤害修复的不同贡献。该提议的目的旨在 解决干细胞的特定贡献及其直接对角膜再生的后代并进行测试 它们需要伤口愈合的必要性。目的我还将解决关键分子信号在调节命运中的作用 角膜内不同的上皮种群。此类信号可能被利用用于治疗 目的。 AIM II将研究角膜干细胞的外部调节。基础神经丛是一个 角膜组织环境的主要组成部分。我们的现场成像方法是研究 角膜神经与上皮之间的功能相互作用。我们已经设计了体内测定 可视化和操纵活角膜中的神经过程。 AIM II将测试子基的需求 神经丛，用于稳态和伤口愈合的上皮功能的神经丛，并探索新的机制 角膜神经再生。这些数据将为神经性角膜炎患者提供新的疗法。这 研究具有创新性，因为它使用尖端的成像技术和遗传工具来研究干细胞 在活哺乳动物眼的自然组织环境中。我们的目标是揭示基本 角膜再生的机制将释放干细胞治疗角膜疾病的全部潜力。