Characterization of Epidermal Stem Cells

描述（由申请人提供）：该项目的长期目标是在已知干细胞含量的相对纯净的群体中获得表皮干细胞，以便为伤口愈合、干细胞靶向基因治疗提供改进的治疗方法，并更好地了解表皮分化和表皮癌变。在目标1中，我们将首先描述滤泡与滤泡间表皮干细胞的数量、多能性和关系。具体来说，我们将在体内竞争性再生实验中使用限制稀释分析来确定滤泡干细胞和滤泡间干细胞的数量。通过分离细胞群（滤泡vs滤泡间）和测定GFP标记的干细胞及其后代在一次移植室中产生滤泡间表皮的能力，然后在随后的二次移植室中产生滤泡或其部分的能力，来确定滤泡干细胞与滤泡间干细胞的多能性。在Aim 2中，进一步的研究将使用既建立的富集方法，以及新的选择策略组合，来确定最有效的表皮干细胞选择策略，以长期再生表皮细胞（干细胞）。每个潜在的单独分离技术将通过执行角蛋白细胞富集群体的限制稀释研究进行测试。然后，我们将确定阳性和阴性标记的最佳组合，以有效地分离表皮干细胞。这种高度富集的干细胞群体将用于使用微阵列分析确定表皮干细胞特异性基因转录。最后，在Aim 3中，我们将使用过表达HOX B4或sonic hedgehog这两种与其他器官中干细胞更新增加相关的蛋白来扩增表皮干细胞群。这些研究将有助于阐明滤泡干细胞和滤泡间干细胞之间的关系及其多能性程度，并进一步分离出真正的表皮长期再生细胞。丰富的表皮干细胞群体将有助于干细胞导向的基因治疗，并且当扩增时将作为潜在的伤口/烧伤愈合治疗。

DESCRIPTION (provided by applicant): The long-term objective of this project is to obtain epidermal stem cells in a relatively pure population of known stem cell content in order to provide improved therapy for healing of wounds, for stem cell-targeted gene therapy, and to better understand epidermal differentiation and epidermal carcinogenesis. In Aim 1, we will first characterize the number, multipotency, and relationship of follicular vs. interfollicular epidermal stem cells. Specifically, we will determine the number of follicular vs. interfollicular stem cells, using limiting dilution analysis in an in vivo competitive repopulation assay. The multipotency of follicular vs. interfollicular stem cells will then be determined by using isolated cell populations (follicular vs. interfollicular) and determining the ability of a GFP marked stem cell and its progeny to produce interfollicular epidermis in a primary transplantation chamber, followed by a follicle or part thereof in a subsequent secondary transplantation chamber. In Aim 2 further studies using both established enrichment methods, as well as new combinations of selection strategies, will be used to determine the most effective epidermal stem cell selection strategy for long-term repopulating cells (stem cells) of the epidermis. Each potential individual isolation technique will be tested by performing a limiting dilution study of the enriched population of kertinocytes. We will then determine the best combination of positive and negative markers to effectively isolate epidermal stem cells. This population of highly enriched stem cells will be used to determine epidermal stem cell-specific gene transcription using microarray analysis. Finally, in Aim 3 we will expand an epidermal stem cell population using over-expression of HOX B4 or sonic hedghog, two proteins associated with increased stem cell renewal in other organs.These studies will help clarify the relationship between follicular and interfollicular stem cells and their degree of multipotency, as well as move closer to isolating true long-term repopulating cells of the epidermis. Enriched populations of epidermal stem cells will aid stem cell-directed gene therapy, and when expanded will serve as a potential wound/burn healing therapy.