Genetic mapping of the inflammatory adaption circuit in epithelial stem cells

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.