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.

蝾螈，像蝾螈和美西蝾螈一样，在受伤时可以再生眼睛组织。这可能是由于 他们的色素上皮细胞重新编程为视网膜和透镜的能力。本研究的目标是 解开重编程机制并将其用于再生医学应用。主要 研究的焦点是虹膜色素上皮（IPE）细胞被激活、增殖 重新编程，并最终在完全移除后再生透镜。在蝾螈中，这个过程 仅限于IPE细胞居住在背侧虹膜，而腹侧IPE细胞被认为不参与在 细胞水平。相比之下，幼年蝾螈不能再生它们的透镜。这些再生差异 使实验范式的使用，可以提供重要的见解，分子 IPE重编程机制。在具体目标1中，比较转录组学、流 细胞计数分选和谱系追踪将用于鉴定和表征IPE亚群 引发再生反应的可能性这些方法也将与一种新的转基因蝾螈配对 能够追踪重编程期间眼转录因子动态的变化。这将使我们 以更好地了解再生过程中分子和细胞变化之间的联系。在 具体目标2，将进行透镜再生的功能表征。成功 为了实现这一目标，将实施鸟类RCAS/TVA系统，以提供空间和时间控制 目的基因在色素上皮中的表达。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，使其对RCAS敏感 逆转录病毒使用这个系统，基因和途径的影响将被测试其诱导的能力。 分别抑制腹侧IPE细胞的再生或抑制背侧IPE细胞的再生。这种方法 通过执行必要性和充分性实验提供功能读出。具体目标3中， 老化，DNA损伤和再生潜力之间的关系将被探讨。蝾螈可以 从IPE细胞再生他们的透镜，并且这种能力似乎不受衰老的影响， 反复受伤。首先，将开发一种重复损伤模型，并结合分子标志物， 衰老和DNA损伤。然后，将使用药理学和遗传学方法来研究 对色素上皮细胞重编程为眼组织的能力的遗传毒性应激升高。