Epidermal Stem Cell Contributions to Wound Healing: Unraveling the complex interplay between LGR5 stem cells and WNT signaling

Project Abstract In the skin, the hair follicle stem cells are found within the root of the hair follicle. These cells?marked by the membrane receptor LGR5?remain within their niche in the hair follicle and proliferate to control hair cycle and development. The only time that LGR5+ stem cells or their progeny are found to leave the hair follicle niche is when there is a cutaneous wound in the vicinity, in which case they are shown to contribute to the new wound epithelium. The factors controlling this process are mostly unknown. In this proposal, we seek to clarify the interactions between LGR5 cells and Wnt signaling with the long-term goal of developing therapeutics to treat non-healing wounds. To study LGR5 cells, we have generated a novel transgenic pig model that expresses H2B- GFP under the control of the endogenous LGR5 promoter (LGR5-H2B-GFP). Pigs make an excellent model for studying cutaneous wounding because, in contrast to mice and analogous to humans, they have tight skin attachment, sparse hair coat, thick skin, and independently cycling hair follicles. Using this unique LGR5-H2B- GFP model, we will study the timing and source of LGR5+ cells in response to Wnt signaling. The receptor LGR5 is a known potentiator of Wnt signaling, and LGR5+ cells rely on Wnt signaling during tissue homeostasis, as well as proliferation and migration during hair cycling and growth. This Wnt signaling is complex; it can act through three very different downstream pathways depending on which of the 19 known Wnt genes are expressed. Our goal is to use this novel large animal model to unravel the spatial and temporal interactions between LGR5 stem cells and Wnt signaling during wound repair. We hypothesize that LGR5+ stem cells at the wound edge are induced to proliferate and migrate in response to Wnt signaling from the wound. To test this, we have developed a full thickness porcine wound model. By taking biopsies from the wound edge and wound bed at continuous time points post wounding, we aim to 1) identify and quantify pattern of LGR5+ stem cell response to skin wounding over time and 2) determine composition and location of Wnt signaling over time. With the aims achieved in this proposal, we will establish basic information about the natural process of wound healing in a novel, physiologically relevant animal model, which then can be applied toward development of novel therapeutics for human dysregulated wound healing.