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: 10738453
• 负责人: Konstantinos Sousounis
• 金额: $ 24.81万
• 依托单位: UNIVERSITY OF NEW HAMPSHIRE
• 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 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10738453
• 关键词: Adolescent; Affect; Age; Age related macular degeneration; Aging; Ambystoma; American; Birds; Blindness; Cell Cycle; Cell Cycle Progression; Cell Separation; Cell Survival; Cells; Coupled; Couples; Cre-LoxP; DNA Damage; Data; Diabetic Retinopathy; Disease; Dorsal; Epithelial Cells; Event; Excision; Experimental Designs; Eye; Eye Injuries; Eye diseases; Flow Cytometry; Future; Gene Expression; Gene Transfer Techniques; Genes; Genetic; Genotoxic Stress; Glaucoma; Goals; Human; Immunohistochemistry; Injury; Iris; Life; Location; Mammals; Mentors; Methodology; Methods; Modeling; Molecular; Natural regeneration; Newts; Pathway interactions; Patients; Phase; Pigment Epithelium; Population; Predisposition; Process; Proliferating; Quantitative Reverse Transcriptase PCR; Regenerative Medicine; Regenerative response; Regulation; Rejuvenation; Research; Retina; Retroviridae; Risk Factors; Role; Salamander; Sorting; Source; System; Testing; Therapeutic; Time; Tissues; Transgenes; Transgenic Organisms; Translating; Tva receptor; Virus; Vision; Western Blotting; comparative; experimental study; genetic manipulation; improved; in vivo; injured; insight; lens; lens regeneration; novel; pharmacologic; progenitor; programs; promoter; protein protein interaction; regeneration potential; regenerative; repaired; response; single-cell RNA sequencing; spatiotemporal; stem; stem cell proliferation; stem cells; tissue regeneration; transcription factor; transcriptomics; vector

Salamanders, like newts and axolotls, can regenerate ocular tissues when injured. This is possible due to the ability of their pigmented epithelial cells to reprogram to retina and lens. The goal of this research is to unravel the reprogramming mechanism and leverage it for regenerative medicine applications. The main focus of the study is the ability of iris pigmented epithelial (IPE) cells to get activated, proliferate, reprogram, and ultimately regenerate the lens upon its complete removal. In newts, this process is restricted to IPE cells residing in the dorsal iris while ventral IPE cells are thought to not participate at the cellular level. In contrast, juvenile axolotls cannot regenerate their lens. These regenerative discrepancies enable the use of experimental paradigms that could provide important insights into the molecular mechanism of IPE reprogramming. In Specific Aim 1, a combination of comparative transcriptomics, flow cytometry sorting and lineage tracing will be used to identify and characterize IPE subpopulations capable of triggering a regenerative response. These approaches will also be paired with a novel transgenic newt capable of tracking changes in eye transcriptional factor dynamics during reprogramming. This will allow us to better understand the association between molecular and cellular changes during regeneration. In Specific Aim 2, functional characterization of lens regeneration will be performed. To successfully accomplish that, the avian RCAS/TVA system will be implemented to provide spatial and temporal control of target gene expression in the pigmented epithelium. The avian TVA receptor will be ubiquitously expressed in eye tissues, in dorsal IPE cells, or in ventral IPE cells making them susceptible to the RCAS retrovirus. Using this system, the effects of genes and pathways will be tested for their ability to induce regeneration from the ventral, or inhibit regeneration from the dorsal IPE cells, respectively. This approach provides a functional readout by performing necessity and sufficiency experiments. In Specific Aim 3, the relationship between aging, DNA damage, and regeneration potential will be explored. Newts can regenerate their lens from IPE cells throughout life and the ability appears unaffected by aging and repeated injury. First, a repeated injury model will be developed and coupled with molecular hallmarks of aging and DNA damage. Then, pharmacological and genetic methods will be used to study the effects of elevated genotoxic stress on the ability of the pigmented epithelium to reprogram to eye tissues.

Salamanders，例如Newts和Axolotls，在受伤时会再生眼组织。这可能是由于 它们有色上皮细胞重新编程为视网膜和镜头的能力。这项研究的目的是 揭示重编程机制并利用其用于再生医学应用。主 该研究的重点是虹膜色素上皮（IPE）细胞的能力被激活，增殖， 重新编程，并在完全去除镜头后最终再生。在newts中，这个过程是 仅限于居住在背虹膜中的IPE细胞，而腹侧IPE细胞被认为不参与 细胞水平。相反，青少年的Axolotls无法再生其镜头。这些再生差异 启用实验范式，可以为分子提供重要的见解 IPE重新编程的机制。在特定的目标1中，比较转录组学的组合，流量 细胞仪分类和谱系跟踪将用于识别和表征IPE亚群 触发再生响应。这些方法也将与新型的转基因纽特配对 能够跟踪重编程过程中眼部转录因子动力学的变化。这将使我们 更好地了解再生过程中分子变化和细胞变化之间的关联。在 将执行特定目标2，透镜再生的功能表征。成功 这样一来，将实施禽类/TVA系统以提供空间和时间控制 靶基因表达在色素上皮中。鸟类TVA受体将无处不在 在眼组织，背侧IPE细胞或腹侧IPE细胞中表达，使其容易受到RCA的影响 逆转录病毒。使用该系统，将测试基因和途径的效果，以诱导它们的能力 腹侧再生或分别抑制背侧IPE细胞的再生。这种方法 通过执行必要性和充分性实验来提供功能读数。在特定的目标3中 将探索衰老，DNA损伤和再生潜力之间的关系。纽特可以 一生中从IPE细胞中再生其镜头，并且能力似乎不受衰老和 反复受伤。首先，将开发出重复的伤害模型，并与分子标志结合 衰老和DNA损伤。然后，将使用药理学和遗传方法来研究 对色素上皮对眼组织重新编程的能力的遗传毒性应激升高。