Age-Related Changes in Epidermal Stem Cells

在过去，申请人的研究集中在衰老、表皮脂质和表皮渗透性屏障上。 这项申请反映了PI方向的重大变化，其长期目标是研究衰老对表皮干细胞的影响。 短期目标是优化一种新的角质形成干细胞体内竞争性再增殖试验，以研究干细胞的老化。表皮干细胞的研究一直受到阻碍，缺乏标志物来识别他们，这反过来又受到缺乏足够的体内干细胞测定的限制。 对表皮干细胞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再生细胞。借鉴成熟的造血细胞功能检测方法，我们设计了一种新的体内竞争性再生检测方法来研究表皮细胞中干细胞的功能。 我们的测定利用绿色荧光蛋白（GFP）区分表皮来源的测试细胞与非绿色竞争细胞。当接种在非GFP小鼠背部的腔室中时，这些角质形成细胞产生表皮。有限稀释技术允许使用该测定法确定角质形成细胞群体中干细胞的绝对数量。 该测定具有两个关键特征。 第一种是使用长期竞争性测定法，其不仅确保检测到最原始的表皮干细胞，而且确保移植的角质形成细胞的存活，即使当用非常低数量的测试细胞接种时，而且确保移植的角质形成细胞的存活，即使当用非常低数量的测试细胞接种时。 第二种是使用有限稀释设计来允许干细胞定量。这些研究的目的是首先充分优化我们的测定，使我们能够评估老年和年轻的表皮干细胞。 然后，我们将比较来自年轻与老年表皮的干细胞的绝对数量。这些研究将在鼠角质形成细胞以及人角质形成细胞中进行。这项工作将使我们开始了解表皮干细胞随年龄的变化，并可能指向进一步的研究，以改善表皮细胞功能和/或伤口愈合的老年人。这些研究还将提供第一个表皮干细胞的体内检测，这是分析这种难以捉摸的表皮细胞的推定标记物，皮肤干细胞靶向基因治疗以及表皮干细胞调控和分化的更多基础研究的重要一步。

In the past, the applicant's research has focused on aging, epidermal lipids, and the epidermal permeability barrier. This application reflects a significant change in direction for the PI, whose long-term objective is to study the effects of aging on epidermal stem cells. The short-term objective is to optimize a novel in vivo competitive repopulation assay for keratinocyte stem cells, in order to study the aging of stem cells. Studies of epidermal stem cells have been hampered by lack of markers by which to identify them, which in turn has been limited by lack of adequate in vivo stem cell assays. Studies of epidermal stem cells performed to 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 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. The aim of these studies is first to fully optimize our assay, to enable us to assess aged and young epidermal stem cells. We will then compare the absolute number of stem cells from young vs. aged epidermis. These studies will be done in murine keratinocytes as well as in human keratinocytes. 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 in vivo assay for epidermal stem cells, an important step for analyzing 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.