Epidermal Stem Cells: Age-Related Changes

描述(申请人提供)：表皮干细胞很有潜力 重要是因为它们在组织动态平衡、伤口修复等方面发挥着核心作用 (两者都延迟衰老)，以及在肿瘤启动和基因治疗方面。 由于缺乏标记物，表皮干细胞的研究一直受到阻碍 用什么来识别他们。对推定标记的研究反过来也是有限的。 由于缺乏足够的功能性干细胞分析。表皮干细胞的研究进展 到目前为止进行的细胞培养依赖于相对短期的体外集落形成 单位作为评估茎的工具。然而，对其他组织的研究已经 表明体外集落形成单位并不能代表真正的长期 正在重新填充细胞。 借用成熟的造血细胞功能分析，我们有 设计了一种新的体内竞争再繁殖实验来研究干细胞 在表皮细胞中发挥作用。我们的检测使用绿色荧光蛋白(GFP) 以区分表皮来源的测试细胞和非绿色竞争细胞。 这些角质形成细胞种植在背部的小室中时会产生一层表皮。 非绿色荧光蛋白小鼠。限制稀释技术允许绝对数量的 角质形成细胞群体中的干细胞将使用这一检测方法来确定。这 化验有两个关键特征。第一个是使用长期竞争性的 这种方法不仅能检测到最原始的表皮干细胞 细胞和移植角质形成细胞的存活，即使在种子 测试电池的数量非常少。第二种是限制的使用。 稀释设计，允许干细胞量化。 我们的长期目标是阐明衰老对表皮干细胞的影响 细胞，并确定延迟表皮形成的机制(S) 动态平衡和伤口愈合可能会得到改善。具体目标包括， 第一，充分优化我们的检测方法，使我们能够评估老少 小鼠表皮干细胞。然后我们将比较词干的绝对数量 细胞的数量，并确定其增殖潜能。 来自年轻和老年表皮的相同数量的干细胞。 这项工作将使我们开始了解表皮干细胞的变化 随着年龄的增长，细胞的数量增加，这可能表明进一步研究如何改善表皮细胞 老年人的功能和/或伤口愈合。这些研究还将提供 第一个有功能的体内表皮干细胞测试，这是 分析(这种难以捉摸的表皮细胞的T假定标志物，用于皮肤 干细胞靶向基因治疗，以及更多关于表皮干细胞的基础研究 细胞的调节和分化。

DESCRIPTION (provided by applicant): Epidermal stem cells are of great importance because they play a central role in tissue homeostasis, wound repair (both delayed in aging), as well as in tumor initiation, and for gene therapy. Studies of epidermal stem cells have been hampered by a lack of markers by which to identify them. Studies of putative markers have in turn been limited by lack of adequate functional stem cell assays. Studies of epidermal stem cells performed to date rely on relatively short-term in vitro colony forming units as a tool to assess ?stemness?. However, studies in other tissues have shown that in vitro colony forming units do not represent the true long-term repopulating cell. Borrowing from well-established functional assays in hemopoietic cells, we have designed a novel in vivo competitive repopulating assay to study stem cell function in epidermal cells. Our assay utilizes green fluorescent protein (GFP) to distinguish epidermally derived test cells from non-green competitor cells. These keratinocytes produce an epidermis when seeded in a chamber on the back of a non-GFP mouse. A limiting dilution technique allows the absolute number of stem cells in a keratinocyte population to be determined using this assay. This assay has two key features. The first is the use of a long-term competitive assay that ensures not only the detection of the most primitive epidermal stem cells but also the survival of the transplanted keratinocytes, even when seeded with very low numbers of test cells. The second is the use of a limiting dilution design to allow stem cell quantification. Our long-term objective is to elucidate the effects of aging on epidermal stem cells, and determine the mechanism(s) by which the delayed epidermal homeostasis and wound healing may be improved. The specific aims include, first, to fully optimize our assay, to enable us to assess aged and young murine epidermal stem cells. We will then compare the absolute number of stem cells in aged vs. young epidermis, and determine the proliferative potential of an equal number of stem cells from young vs. aged epidermis. This work will allow us to begin to understand the changes in epidermal stem cells with age, and may point to further studies to improve epidermal cell function and/or wound healing in the aged. These studies will also provide the first functional in vivo assay for epidermal stem cells, an important step for analvzing (T putative markers for this elusive epidermal cell, for cutaneous stem cell-targeted gene therapy, and for more basic studies of epidermal stem cell regulation and differentiation.