Characterization of Epidermal Stem Cells

描述(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人群中获得表皮干细胞，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，我们将首先描述毛囊和毛囊间表皮干细胞的数量、多潜能以及它们之间的关系。具体地说，我们将在体内竞争性再繁殖试验中使用有限稀释分析来确定卵泡干细胞与卵泡间干细胞的数量。然后，将通过使用分离的细胞群(卵泡和卵泡间)，并确定GFP标记的干细胞及其后代在一次移植腔中产生卵泡间表皮的能力，然后在随后的二次移植腔中产生一个卵泡或其中的一部分，来确定滤泡和卵泡间干细胞的多能性。在目标2中，将使用已建立的浓缩方法以及新的选择策略组合进行进一步的研究，以确定长期重新填充表皮细胞(干细胞)的最有效的表皮干细胞选择策略。每种可能的个体分离技术都将通过对浓缩的角质细胞群体进行有限稀释研究来测试。然后，我们将确定有效分离表皮干细胞的阳性和阴性标记的最佳组合。这种高度浓缩的干细胞群体将被用于通过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，我们将通过过量表达Hox B4或sonic hedghog来扩大表皮干细胞种群，这两种蛋白质与其他组织中干细胞更新的增加有关。这些研究将有助于澄清毛囊和毛囊间干细胞之间的关系及其多能性程度，以及更接近分离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，当扩增时，将作为一种潜在的伤口/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.