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.
