Elucidation of cellular reprogramming processes that drive lens regeneration in axolotl as a basis for future therapeutic approaches

阐明驱动蝾螈晶状体再生的细胞重编程过程，作为未来治疗方法的基础

• 批准号: 9918425
• 负责人: Konstantinos Sousounis
• 金额: $ 12.86万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9918425
• 关键词: Adult; Affect; Age related macular degeneration; Ambystoma; American; Amphibia; Antibodies; Award; Blindness; Candidate Disease Gene; Cell Separation; Cell Survival; Cell physiology; Cells; Chemicals; Clinical; DNA Damage; DNA Repair; Data; Data Collection; Development; Diabetic Retinopathy; Disease; Educational workshop; Epithelial Cells; Event; Excision; Experimental Designs; Eye; Eye diseases; Family; Feedback; Fluorescence; Foundations; Future; Gene Expression; Genetic; Genotoxic Stress; Glaucoma; Goals; Gold; Heterogeneity; Hospitals; Human; Injury; Intervention; Iris; Journals; Knock-in; Knock-out; Laboratories; Laboratory Research; Larva; Lead; Leadership; Learning; Lens Placodes; Link; Location; Mammals; Mediating; Medical; Mentors; Methodology; Modeling; Modernization; Molecular; Mus; Natural regeneration; Newts; Organ; Organism; Pathway interactions; Patients; Pharmaceutical Preparations; Phase; Phosphoric Monoester Hydrolases; Pigment Epithelium; Pigments; Population; Process; Proteins; Regenerative Medicine; Regenerative response; Research; Retina; Role; Salamander; Scientist; Seeds; Source; Speed; System; Testing; Therapeutic; Time; Tissues; Training; Transgenic Animals; Transgenic Organisms; Translating; Vertebrates; Vision; Visualization; Woman; age related; base; blind; career; experience; experimental study; genomic locus; hatching; human stem cells; improved; in silico; in vivo; insight; lens; lens regeneration; medical schools; meetings; novel; progenitor; recombinase; regenerative; restoration; senescence; single-cell RNA sequencing; skills; small molecule; small molecule libraries; stem; stem cell proliferation; stem cells; tissue processing; tissue regeneration; tool; transcription factor; transcriptomics

The goal of the project is to harness the intrinsic ability of axolotl salamanders to regenerate ocular tissues and unravel natural ways to improve human stem cell function for use in regenerative medicine. Axolotl’s ability to regenerate the retina and the lens stems from the pigmented epithelium, which reprograms its cellular state upon injury. To understand the mechanism of regeneration I will focus on iris pigmented epithelial (IPE) cells and their capacity to reprogram to lens epithelial cells. This process is age-dependent in axolotls because regeneration can only occur for a short time window of two weeks after hatching and is lost thereafter. This allows me to both identify and functionally characterize the potency of candidate molecules to induce regenerative responses in the same context. Studying axolotl IPE reprogramming will provide important insight into how cellular plasticity has naturally evolved in organisms as a mean to regenerate lost organs. Modern regenerative medicine can learn from this intrinsic regenerative mechanism and apply it to coax reprogramming of any adult cell in a safer and more efficient way. In addition, its application could lead to groundbreaking treatments for blind patients by inducing ocular regeneration. In Specific Aim 1, I will study the role of Eya2 during IPE reprogramming. My preliminary data indicate that Eya2 is required for progenitor cell survival upon genotoxic stress during regeneration. I will also study Eya2’s ability to facilitate IPE reprogramming by regulating the activity of transcription factors required to maintain specific eye-tissue fates. In Specific Aim 2, I will generate a transgenic axolotl model that will allow tracking of lens regeneration by fluorescence in vivo. This transgenic animal will enable me to screen thousands of compounds in a high- throughput manner for their ability to either inhibit or induce regeneration from the IPE. In Specific Aim 3, I will study the cellular heterogeneity of the IPE with single cell RNA-seq. Pseudotime lineage trace analysis will be used to identify whether an IPE-residing progenitor population is present before injury and if it is responsible for regeneration. The in silico identification of a regeneration-poised IPE subpopulation will be further validated and characterized using the gold-standard CreERT2/loxP system in which the recombinase will be targeted in progenitor cell-specific genetic loci. My long-term career goal is to lead a research laboratory with central aim to bridge the regenerative ability gap between salamanders and mammals. My experience working with newts, axolotls and mice, in addition to the data I will acquire during the award training period in the Whited laboratory at Brigham and Women’s Hospital and Harvard Medical School, will enable me to build a foundation for pilot translational experiments in mammals in the near future. My access to and participation in a plethora of meetings, journal clubs, seminars and workshops provide me with invaluable opportunities to communicate my research in academic and clinical settings, receive constructive feedback as well as enhance my professional and leadership skills on my path to becoming a successfully independent scientist.

该项目的目标是利用蝾螈的内在能力来再生眼组织， 揭示自然的方法来改善人类干细胞功能，用于再生医学。美西蝾螈的能力 再生视网膜和透镜源于色素上皮，色素上皮重新编程其细胞状态 在受伤时。为了了解再生的机制，我将重点放在虹膜色素上皮（IPE）细胞 以及它们重编程为透镜上皮细胞的能力。蝾螈的这一过程与年龄有关， 再生只能在孵化后两周的短时间窗口内发生，此后就消失了。这 使我能够识别和功能性地表征候选分子诱导 在相同的背景下进行再生反应。研究蝾螈IPE重编程将提供重要的见解 细胞可塑性是如何在生物体中自然进化的，作为再生失去的器官的一种手段。现代 再生医学可以从这种内在的再生机制中学习， 以更安全、更有效的方式对任何成年细胞进行重编程。此外，其应用可能导致 通过诱导眼部再生为盲人提供开创性的治疗。在具体目标1中，我将研究 Eya 2在IPE重编程过程中的作用。我的初步数据表明，Eya 2是祖细胞 在再生过程中遗传毒性胁迫下的存活。我还将研究Eya 2促进IPE的能力 通过调节维持特定眼组织命运所需的转录因子的活性来重编程。 在具体目标2，我将产生一个转基因蝾螈模型，将允许跟踪透镜再生， 体内荧光。这种转基因动物将使我能够筛选数千种化合物在一个高- 它们抑制或诱导IPE再生的能力。在第三个目标中，我将 用单细胞RNA-seq研究IPE的细胞异质性。伪时间谱系跟踪分析将是 用于确定在受伤前是否存在IPE居住的祖细胞群体，以及是否负责 再生将进一步验证再生平衡IPE亚群的计算机识别 并使用金标准CreERT 2/loxP系统进行表征，其中重组酶将被靶向于 祖细胞特异性遗传位点。我的长期职业目标是领导一个研究实验室， 弥补蝾螈和哺乳动物之间的再生能力差距。我和蝾螈一起工作的经验， 蝾螈和老鼠，除了我将在怀特实验室的奖励培训期间获得的数据外， 在布里格姆妇女医院和哈佛医学院，将使我能够建立一个飞行员的基础， 在不久的将来在哺乳动物中进行转化实验。我接触和参与了大量的 会议、期刊俱乐部、研讨会和讲习班为我提供了宝贵的机会，让我能够交流我的想法。 在学术和临床环境中进行研究，获得建设性的反馈，并提高我的专业水平 和领导才能，帮助我成为一名成功的独立科学家。