Epidermal Stem Cells: Age-Related Changes

描述（由申请人提供）：表皮干细胞具有很大的 重要性是因为它们在组织稳态、伤口修复 (both延缓衰老），以及用于肿瘤起始，以及用于基因治疗。 表皮干细胞的研究一直受到缺乏标记物的阻碍， 来识别他们。对假定的标记的研究反过来又受到限制 缺乏足够的功能性干细胞检测。表皮干的研究 迄今为止进行的细胞培养依赖于相对短期的体外集落形成 单位作为评估工具？sterness？然而，在其他组织中的研究 表明体外集落形成单位并不代表真正的长期 重新填充细胞 借用成熟的造血细胞功能测定，我们有 设计了一种新的体内竞争性再生试验来研究干细胞 在表皮细胞中起作用。我们的检测利用绿色荧光蛋白（GFP） 区分表皮来源的测试细胞与非绿色竞争细胞。 这些角质形成细胞在背部的一个腔室中接种时产生表皮 一只非GFP的老鼠。有限稀释技术允许 角质形成细胞群中的干细胞。这 分析具有两个关键特征。第一种是利用长期的竞争力 该测定不仅确保检测最原始的表皮干 细胞，而且移植的角质形成细胞的存活，甚至当接种时 测试细胞的数量非常少。第二种是使用限制 稀释设计以允许干细胞定量。 我们的长期目标是阐明衰老对表皮干细胞的影响 细胞，并确定机制（S），其中延迟表皮 可以改善体内平衡和伤口愈合。具体目标包括， 首先，充分优化我们的检测，使我们能够评估老年人和年轻人， 小鼠表皮干细胞然后我们将比较茎的绝对数量 细胞在老年与年轻的表皮，并确定增殖潜力 相同数量的干细胞来自年轻和年老的表皮。 这项工作将使我们开始了解表皮干的变化 细胞随着年龄的增长，并可能指向进一步的研究，以改善表皮细胞 功能和/或伤口愈合。这些研究还将提供 第一个表皮干细胞的功能性体内试验， 分析这种难以捉摸的表皮细胞的假定标记， 干细胞靶向基因治疗，并为表皮干细胞的更多基础研究 细胞调节和分化。

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.