Genetic mapping of the inflammatory adaption circuit in epithelial stem cells

项目摘要 上皮干细胞存在于主要的屏障组织中，控制稳态再生和损伤修复。作为 上皮干细胞是长寿且不可或缺的细胞，必须忍受炎症发作。这种能力是 当许多免疫细胞渗入组织时，在伤口愈合期间尤其关键。这些免疫细胞 它们在控制感染和清除死亡细胞方面发挥着重要作用，但它们也会释放有毒物质， 对干细胞来说是一个非常严酷的炎症环境。长期以来，人们一直认为干细胞是脆弱的， 并且必须在“免疫特权”的小生境中受到保护。然而，我们最近的研究挑战了这个想法。我们 已经发现，在受伤时，上皮干细胞必须被动员以离开它们的自然生态位并迁移 进入高度炎症的创伤环境，用于再生受损组织。如果干细胞不能 适应炎症，它可能会导致无法愈合的伤口，这仍然影响着全球数百万人， 严重的经济和公共卫生负担。目前尚不清楚上皮干细胞如何实现自我更新， 在炎症环境中的分化，同时防止附带损伤。处理这个问题 将改变我们对细胞适应性、应激耐受性、组织 体内平衡、屏障完整性和伤口修复。由于其重要性，这项建议的核心问题是， 是了解上皮干细胞如何适应炎症环境， 促进伤口修复。技术上的重大差距阻碍了对伤口愈合的全面了解 和干细胞的适应性功能是缺乏有效的工具，快速基因发现和机制研究， 小鼠模型。为了克服这一障碍，在本项目中，我们将采用超声引导的子宫内显微注射 技术，建立一个新的实验框架，快速，功能和机制的研究， 直接在活体小鼠中参与干细胞适应和伤口愈合的基因。我们将利用这个实验性的 框架来部署一个成熟的平台，这将使我们处于一个独特的位置：首先，设计体内CRISPR 筛选平台和干细胞相互作用组传感器，以剖析上皮干细胞如何重塑命运 和周围免疫细胞的活动，以建立一个临时的保护龛，保护干细胞免受 炎性损伤其次，我们将专注于设计一个基于体内扰动序列的框架， 细胞/细胞器标记系统，以确定上皮干细胞如何重新编程其代谢， 炎症总之，该提案有可能揭示关键信息，并为以下方面奠定坚实的基础： 未来的努力，在制定战略，以管理不愈合的伤口。

PROJECT SUMMARY Epithelial stem cells reside in the major barrier tissues, governing homeostatic regeneration and injury repair. As long-lived and indispensable cells, epithelial stem cells must endure bouts of inflammation. This ability is especially critical during wound healing when many immune cells infiltrate the tissue. These immune cells play important roles in controlling infections and clearing dead cells, but they also release toxic substances and create a very harsh inflammatory environment for stem cells. It has long been assumed that stem cells are vulnerable and must be protected within an ‘immune privileged’ niche. However, our recent study challenged this idea. We have found that, upon wounding, the epithelial stem cells must be mobilized to exit their natural niche and migrate into a highly inflammatory wounding environment for regenerating the damaged tissue. If stem cells failed to adapt to inflammation, it could cause nonhealing wounds, which still affect millions of people worldwide, causing significant economic and public health burdens. It is unclear how epithelial stem cells achieve self-renewal and differentiation within an inflammatory environment while preventing collateral damage. Addressing this question will transform our understanding of the fundamental biology underlying cellular fitness, stress tolerance, tissue homeostasis, barrier integrity, and wound repair. Driven by its importance, the central question of this proposal is to understand how epithelial stem cells adapt to the inflammatory environment and how this adaptive function promotes wound repair. A significant gap in technology preventing a thorough understanding of wound healing and stem cell adaptive functions is the lack of effective tools for rapid gene discovery and mechanistic studies in mouse models. To overcome this hurdle, in this project, we will adopt an ultrasound-guided in utero microinjection technique to establish a new experimental framework for rapid, functional, and mechanistic investigation of genes involved in stem cell adaptation and wound healing directly in live mice. We will leverage this experimental framework to deploy a full-fledged platform that will place us in a unique position to: first, design in vivo CRISPR screening platforms and stem cell interactome sensors to dissect how epithelial stem cells can remodel the fate and activities of surrounding immune cells to build a temporary protective niche, shielding stem cells from inflammatory damage. Second, we will focus on devising an in vivo Perturb-seq-based framework and cell/organelle tagging system to identify how epithelial stem cells reprogram their metabolism to tolerate inflammation. In sum, this proposal has the potential to reveal critical information and build a solid foundation for future efforts in developing strategies to manage non-healing wounds.