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

该项目的目标是利用紫杉醇火蜥蜴的内在能力再生眼组织和 揭示用于再生医学的改善人类干细胞功能的自然方法。Axolotl的抗癌能力 再生视网膜和晶状体源于有色上皮，它对细胞状态进行重新编程 在受伤的时候。为了了解再生的机制，我将重点研究虹膜色素上皮(IPE)细胞 以及它们重新编程为晶状体上皮细胞的能力。这一过程在Axolotls中是年龄相关的，因为 再生只能在孵化后两周的短时间内发生，此后就会消失。这 使我能够识别并从功能上表征候选分子诱导 在相同背景下的再生反应。研究Axolotl IPE重新编程将提供重要的启示 细胞可塑性是如何在生物体中自然进化的，作为再生丢失器官的一种手段。现代 再生医学可以借鉴这种内在的再生机制，并将其应用于哄骗 以更安全和更有效的方式对任何成年细胞进行重新编程。此外，它的应用可能会导致 通过诱导眼睛再生对盲人患者进行开创性的治疗。在具体目标1中，我将研究 Eya2在IPE重新编程中的作用。我的初步数据显示，祖细胞需要Eya2 在再生过程中在基因毒性应激下存活。我也会研究EYA2的S促进IPE的能力 通过调节维持特定眼睛组织命运所需的转录因子的活性来重新编程。 在特定的目标2中，我将生成一个转基因Axolotl模型，它将通过以下方式跟踪晶状体再生 体内的荧光。这只转基因动物将使我能够筛选出数千种化合物- 他们抑制或诱导从IPE再生的能力的通过方式。在具体目标3中，我将 用单细胞RNA序列研究IPE的细胞异质性。假时间血统分析将会是 用于识别在受伤前是否存在居住在IPE的祖细胞群体，以及它是否对 再生。再生平衡的IPE亚群的电子鉴定将进一步得到验证 并使用以重组酶为靶点的金标准CreERT2/loxP系统进行鉴定 祖细胞特有的遗传位点。我的长期职业目标是领导一个有中心目标的研究实验室 来弥合火蜥蜴和哺乳动物之间的再生能力差距。我的经验表明，我在与海燕打交道时， Axolotl和MICE，除了我将在怀特实验室的获奖培训期间获得的数据之外 在布里格姆妇女医院和哈佛医学院，将使我能够建立一个基础的试点 在不久的将来在哺乳动物身上进行翻译实验。我接触和参与了过多的 会议、日记俱乐部、研讨会和研讨会为我提供了宝贵的机会来交流我的 在学术和临床环境中进行研究，收到建设性的反馈并提高我的专业水平 和领导技能在我成为一名成功的独立科学家的道路上。

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.