Continuously renewing tissues, such as the epidermis, are populated by a hierarchy of dividing transient amplifying cells, which are maintained by stem cells. Transient amplifying cells divide to maintain the tissue, but they are limited to a finite number of cell divisions before they differentiate and are sloughed. Only the stem cells remain for the life of the tissue. Thus, it is critical to target stem cells when designing gene therapy regimes for genetically inherited diseases, such as epidermolysis bullosa simplex (EBS). Unfortunately, isolating pure epithelial stem cells has been problematic. In this study, we used rapid adherence to collagen type IV to successfully enrich for epidermal stem cells from adult human skin. These preselected stem cells were slow to proliferate, but they ultimately formed large colonies. When recombined with the dermal substrate AlloDerm, the stem cells re-formed a stratified squamous epidermis within 1 week after raising the AlloDerm to the air-liquid interface. These organotypic cultures grew continuously and, even after 6 weeks in culture, they maintained a proliferative basal layer. When transduced with a retroviral LacZ vector, preselected stem cells formed beta-galactosidase-positive clones in submerged and organotypic cultures. Transduced cells showed persistent expression through 12 weeks in organotypic culture, demonstrating the feasibility of using preselected stem cells for gene therapy. Currently, we are developing two models of EBS to test a gene therapy approach, which is based on the premise that EBS stem cells with a mutant keratin (K)14 gene corrected to wild type will have a growth advantage over noncorrected EBS stem cells.