Transduction of a preselected population of human epidermal stem cells: Consequences for gene therapy

Continuously renewing tissues, such as the epidermis, are populated by a hi erarchy of dividing transient amplifying cells, which are maintained by ste m 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. T hus, it is critical to target stem cells when designing gene therapy regime s for genetically inherited diseases, such as epidermolysis bullosa simplex (EBS). Unfortunately, isolating pure epithelial stem cells has been proble matic. In this study, we used rapid adherence to collagen type TV to succes sfully enrich for epidermal stem cells from adult human skin. These presele cted stem cells were slow to proliferate, but they ultimately formed large colonies. When recombined with the dermal substrate AlloDerm, the stem cell s re-formed a stratified squamous epidermis within 1 week after raising the AlloDerm to the air-liquid interface. These organotypic cultures grew cont inuously and, even after 6 weeks in culture, they maintained a proliferativ e basal layer. When transduced with a retroviral LacZ vector, preselected s tem cells formed beta-galactosidase-positive clones in submerged and organo typic cultures. Transduced cells showed persistent expression through 12 we eks in organotypic culture, demonstrating the feasibility of using preselec ted 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 EB S stem cells with a mutant keratin (K)14 gene corrected to wild type will h ave a growth advantage over noncorrected EBS stem cells.