All renewing epithelial tissues are maintained by continuous division of cells in the proliferative population to replace cells in the differentiation compartment that are sloughed into the environment. This mechanism of balancing the rate of cell division with the rate of cell loss is essential for epithelial homeostasis and must be maintained for life. In such tissues, the proliferative population consists of a hierarchy of dividing cells maintained for life. In such tissues, the proliferative population consists of a hierarchy of dividing cells maintained by a small subpopulation of stem cells. The stem cells are self-renewing with cell division producing both stem cells and proliferating daughters, the transit amplifying cells. The transit amplifying cells divide to maintain the tissue, but they are limited to a finite number of cell divisions before they differentiate and are ultimately lost. Only the stem cells remain for the life of the tissue. Thus, it is critical when designing gene therapy regimes for genetically inherited diseases that we target the stem cell population. Unfortunately, to data, isolated epithelial stem cells has been problematic. Previously using tritiated thymidine, we were able to label a subpopulation of slowly cycling basal cells in mouse skin and oral mucosa that we called label- retraining cells (LRCs), which showed the characteristics of stem cells. This tagging method has been used for the last decade to identify stem cells in several other epithelia. Recently, we devised a method that enriches the LRC population from 2% in mouse ear tissue to over 90% in the culture dish, and further we have shown that the selected cells show the growth and repopulation potential expected for epidermal stem cells. In this feasibility project, we will test the potential of using these selected stem cells for long term recombinant gene expression. Although several techniques for expressing recombinant genes in epidermal tissues directly or in epidermal cells in culture have been tried, to date only two studies have reported long term DNA expression. They used an ex vivo approach in which they transduced keratinocytes in culture with a retroviral construct, then transplanted the keratinocytes back to the host. Both studies reported a rapid drop in DNA expression, with less than 1% of the initially transduced cells still expressing the recombinant DNA after two months. This suggests that very few stem cells were transduced. In neither study did the researchers try to enrich for stem cells before transfection. Here, we propose to use our novel enrichment method to first select epidermal stem cells, then to test the hypothesis that only stem cells will give long term recombinant gene expression by transfected these preselected cells with a retroviral vector carrying the LacZ reporter gene or with adeno-associated virus with green fluorescent protein, then use the transfected cells to bioengineer an epidermis and test for long term expression both in organotypic culture and in transplanted skin grafts.
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