Live imaging of stem cell dynamics in cornea regeneration

摘要 根据世界卫生组织的数据，全世界有7000万人因角膜炎而视力受损。 相关疾病和伤害。仅在美国，每年就有超过50，000例角膜移植手术 由于缺乏替代治疗。角膜依赖于常驻干细胞来维持视力， 损伤后再生。导致干细胞偏离其正常活性的条件导致角膜炎。 疾病阐明角膜干细胞的身份和调节机制对于设计新的角膜干细胞治疗方案至关重要。 有效治疗角膜病变。我们目前对干细胞的了解还不完全 在活体角膜中的动力学和调节。一个主要的障碍是无法直接可视化单细胞 在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，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工具的活体成像系统，以1）表征干细胞动力学， 2)鉴定干细胞活性内在和外在调节剂，和3）测试它们对角膜的需求 再生在角膜的活体成像实验中，我们发现自体中的干细胞和祖细胞， 角膜上皮细胞在体内平衡过程中以不同的拓扑模式增殖和分化 和伤口愈合。我们假设角膜由异质性干细胞群组成， 对维持体内平衡和损伤修复有独特的贡献。本提案的目标一旨在 解决干细胞及其直接后代对角膜再生的具体贡献，并测试 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， 角膜内不同的上皮细胞群。这种信号可以潜在地用于治疗 目的目的二研究角膜干细胞的外源性调控。基底下神经丛是一个 角膜组织环境的主要成分。我们的实时成像方法非常适合研究 角膜神经和上皮之间的功能性相互作用。我们已经设计了体内试验， 可视化和操纵活体角膜中的神经过程。目标II将测试基底层的要求 神经丛的上皮功能的稳态和伤口愈合，并探索新的机制， 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 研究是创新的，因为它使用尖端的成像技术和遗传工具来研究干细胞 在活的哺乳动物眼睛的天然组织环境中。我们的目标是揭示 角膜再生的机制，这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.