Live imaging of stem cell dynamics in cornea regeneration

摘要 根据世界卫生组织的数据，全球有7000万人因角膜而视力受损 相关疾病和伤害。仅在美国，每年就有50,000多例角膜移植手术 由于缺乏可替代的治疗方法。角膜依靠常驻干细胞来维持视力和高效 受伤后会再生。导致干细胞偏离正常活动的条件会导致角膜 疾病。阐明角膜干细胞的特性和调节机制对于设计新的 治疗角膜病变的有效方法。我们目前对干细胞的了解还不完全。 活体角膜的动力学和调节。一个主要的障碍是无法直接将单个细胞可视化 角膜再生过程中的活性，以阐明干细胞的确切贡献。为了克服这一点，我们 开创了一种新的方法来可视化和跟踪活着的小鼠完整角膜中的干细胞活动，通过2- 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学工具的实时成像系统，以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因素；3)检测它们对角膜的要求 再生。在角膜的活体成像实验中，我们发现自体角膜中的干细胞和祖细胞 内含角膜上皮在动态平衡期间以不同的拓扑模式增殖和分化 和伤口愈合。我们假设角膜由一组不同种类的干细胞组成， 对维持体内平衡和损伤修复做出了独特的贡献。本提案的目的一是为了 解决干细胞及其直接后代对角膜再生的具体贡献，并测试 它们对伤口愈合的必要性。目的我还将讨论关键分子信号在调节命运中的作用 角膜内不同的上皮群。这样的信号有可能被用于治疗 目的。目的II将研究角膜干细胞的外在调节。基底下神经丛是一种 角膜组织环境的主要成分。我们的实时成像方法是研究 角膜神经和角膜上皮之间的功能相互作用。我们已经设计了体内检测方法 可视化和操纵活体角膜中的神经突起。AIM II将测试分基础的要求 神经丛在动态平衡和创面愈合中的上皮功能及探索新的机制 角膜神经再生。这些数据将为神经性角膜炎患者提供新的治疗方法。这 这项研究具有创新性，因为它使用尖端成像技术和基因工具来研究干细胞 在活体哺乳动物眼睛的自然组织环境中。我们的目标是揭开根本 角膜再生机制将释放干细胞治疗角膜疾病的全部潜力。

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.