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

项目摘要 在皮肤中，毛囊干细胞被发现在毛囊的根部。这些细胞标记为 膜受体LGR 5-保持在毛囊中的小生境内并增殖以控制毛发周期， 发展发现LGR 5+干细胞或其后代离开毛囊龛的唯一时间是 当附近有皮肤伤口时，在这种情况下，它们被证明有助于新的伤口 上皮控制这一过程的因素大多是未知的。在本建议中，我们力求澄清 LGR 5细胞和Wnt信号之间的相互作用，长期目标是开发治疗方法， 无法愈合的伤口为了研究LGR 5细胞，我们已经建立了一种新的转基因猪模型， 在内源性LGR 5启动子的控制下的GFP（LGR 5-H2 B-GFP）。猪是一个很好的模型， 研究皮肤创伤，因为与小鼠相反，与人类相似，它们的皮肤很紧， 附着、被毛稀疏、皮肤厚、毛囊独立循环。使用这种独特的LGR 5-H2 B- 在GFP模型中，我们将研究LGR 5+细胞响应Wnt信号传导的时间和来源。受体LGR 5 是Wnt信号传导的已知增强剂，并且LGR 5+细胞在组织稳态期间依赖于Wnt信号传导，如 以及毛发周期和生长期间的增殖和迁移。这种Wnt信号是复杂的，它可以作用于 通过三个非常不同的下游途径，这取决于19个已知的Wnt基因中的哪一个是 表达。我们的目标是使用这种新型的大型动物模型来解开空间和时间的相互作用 LGR 5干细胞和Wnt信号在伤口修复过程中的作用。我们假设LGR 5+干细胞在 伤口边缘响应于来自伤口的Wnt信号传导而被诱导增殖和迁移。为了验证这个， 我们已经开发了全厚度的猪伤口模型。通过从伤口边缘和伤口上取活检 在创伤后的连续时间点，我们的目标是1）鉴定和量化LGR 5+干细胞的模式， 随时间推移对皮肤创伤的反应和2）随时间推移确定Wnt信号传导的组成和位置。与 为了达到这个目标，我们将建立有关伤口愈合的自然过程的基本信息。 在新的生理学相关的动物模型中，然后可以将其应用于开发新的 用于人类失调的伤口愈合的治疗剂。

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.