EVIDENCE FOR KERATINOCYTE STEM-CELLS IN-VITRO - LONG-TERM ENGRAFTMENTAND PERSISTENCE OF TRANSGENE EXPRESSION FROM RETROVIRUS-TRANSDUCED KERATINOCYTES

Epidermis is renewed by a population of stem cells that have been defi ned in vivo by slow turnover, label retention, position in the epiderm is, and enrichment in beta(1) integrin, and in vitro by clonogenic gro wth, prolonged serial passage, and rapid adherence to extracellular ma trix. The goal of this study is to determine whether clonogenic cells with long-term growth potential in vitro persist in vivo and give rise to a fully differentiated epidermis, Human keratinocytes were genetic ally labeled in culture by transduction with a retrovirus encoding the lacZ gene and grafted to athymic mice. Analysis of the cultures befor e grafting showed that 21.1-27.8% of clonogenic cells with the capacit y for >30 generations were successfully transduced, In vivo, beta-gala ctosidase (beta-gal) positive cells participated in the formation of a fully differentiated epithelium and were detected throughout the 40-w eek postgraft period, initially as loosely scattered clusters and late r as distinct vertical columns. Viable cells recovered from excised gr afts were seeded at clonal densities and 23.3-33.3% of the colonies th us formed were beta-gal positive. In addition, no evidence of transgen e inactivation mas obtained: all keratinocyte colonies recovered from grafted tissue that were beta-gal negative also tacked the lacZ transg ene. These results show that cells with long-term growth properties in vitro do indeed persist in vive and form a fully differentiated epide rmis, thereby exhibiting the properties of stem cells.