Genetic mapping of the inflammatory adaption circuit in epithelial stem cells

项目总结 上皮干细胞存在于主要的屏障组织中，控制体内平衡再生和损伤修复。AS 上皮干细胞是长寿的不可或缺的细胞，必须经受住一阵阵的炎症。这种能力是 尤其是在伤口愈合过程中，当许多免疫细胞渗透到组织中时。这些免疫细胞扮演着 在控制感染和清除死亡细胞方面发挥重要作用，但它们也释放有毒物质并产生 干细胞所处的炎性环境非常恶劣。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”的利基范围内受到保护。然而，我们最近的研究对这一观点提出了质疑。我们 已经发现，一旦受伤，上皮干细胞必须被动员起来，离开它们的自然生态位并迁移 进入一个高度炎症的创伤环境，以再生受损的组织。如果干细胞不能 适应炎症，它可能会导致无法愈合的伤口，这种伤口仍然影响着全球数百万人，导致 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和 在炎性环境中实现差异化，同时防止附带损害。回答这个问题 将改变我们对细胞健康、压力耐受性、组织 动态平衡、屏障完整性和伤口修复。由于其重要性，这项提案的核心问题 是了解上皮干细胞如何适应炎症环境以及这种适应功能是如何发挥作用的 促进伤口修复。技术上的重大差距阻碍了对伤口愈合的透彻了解 和干细胞适应功能是缺乏有效的工具来快速发现基因和机制研究 老鼠模型。为了克服这一障碍，在这个项目中，我们将采用超声引导的宫内显微注射 为快速、功能性和机械性研究建立新的实验框架的技术 直接在活体小鼠体内参与干细胞适应和伤口愈合的基因。我们将利用这一实验 部署一个成熟的平台的框架，这将使我们处于独特的地位：第一，在vivo CRISPR中设计 筛选平台和干细胞相互作用组传感器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性利基，保护干细胞免受 炎性损伤。第二，我们将专注于设计一个基于体内扰动序列的框架和 细胞/细胞器标记系统识别上皮干细胞如何重新编程其新陈代谢以耐受 发炎。总而言之，这项建议有可能揭示关键信息并为 未来在制定管理非愈合伤口的战略方面的努力。

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.