Selection and extended growth of murine epidermal stem cells in culture

Continuously renewing epithelia contain small undifferentiated stem cells capable of self-renewal and maintenance of the differentiating cell population. In murine epidermis stem cells have been identified as label-retaining cells (LRCs) by long-term retention of tritiated thymidine or BrdU. It has been suggested that epidermal stem cells adhere to basement membranes through differential expression of specific integrins. To determine whether we could use a specific integrin to enrich for murine epidermal stem cells, we tested adherence of LRCs to several substrates. Regardless of the substrate used, approximately 10% of total basal cells and 100% of LRCs adhered in 10 min. In our medium specifically formulated for murine keratinocytes, rapidly adherent stem cells formed large colonies and could be used to form a structurally complete epidermis in organotypic culture. They showed a fivefold greater transient transfection efficiency than total basal cells, and when individual adherent cells were transduced with a retroviral vector, they formed large clones. Although these stem cells grew more slowly than the total basal cell population, they could be subcultured more times. Our results indicate that murine epidermal stem cells can be selected by rapid attachment to a substrate, but not by one specific integrin, and that they can be expanded in culture if the appropriate conditions are maintained.