Characterization of Epidermal Stem Cells

描述（由申请人提供）：本项目的长期目标是获得已知干细胞含量的相对纯的表皮干细胞群，以便为伤口愈合、干细胞靶向基因治疗提供改进的治疗，并更好地了解表皮分化和表皮癌发生。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。具体而言，我们将在体内竞争性再增殖试验中使用有限稀释分析来确定滤泡干细胞与滤泡间干细胞的数量。然后通过使用分离的细胞群（滤泡干细胞对滤泡间干细胞）并确定GFP标记的干细胞及其后代在初次移植室中产生滤泡间表皮，随后在随后的二次移植室中产生滤泡或其部分的能力，来确定滤泡干细胞对滤泡间干细胞的多能性。在目标2中，使用已建立的富集方法以及选择策略的新组合的进一步研究将用于确定用于表皮的长期再增殖细胞（干细胞）的最有效的表皮干细胞选择策略。将通过对角膜细胞富集群体进行有限稀释研究，对每种潜在的个体分离技术进行检测。然后，我们将确定阳性和阴性标记物的最佳组合，以有效分离表皮干细胞。该高度富集的干细胞群体将用于使用微阵列分析来确定表皮干细胞特异性基因转录。最后，在目标3中，我们将使用HOX B4或sonic hedghog的过表达来扩增表皮干细胞群体，这两种蛋白与其他器官中干细胞更新的增加有关。这些研究将有助于澄清滤泡和滤泡间干细胞之间的关系及其多能性程度，并更接近于分离表皮的真正长期再生细胞。表皮干细胞的富集群体将有助于干细胞定向基因治疗，并且当扩展时将用作潜在的伤口/烧伤愈合疗法。

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.