Continuous renewal of the epidermis and its appendages throughout life depends on the proliferation of a distinct population of cells called stem cells. We have used in situ retrovirus-mediated gene transfer to genetically mark cutaneous epithelial stem cells of adolescent mice, and have followed the fate of the marked progeny after at least 37 epidermal turnovers and ®ve cycles of depilation-induced hair growth. Histological examination of serial sections of labeled pilosebaceous units demonstrated a complex cell lineage. In most instances, labeled cells were con®ned to one or more follicular compartments or solely to sebaceous glands. Labeled keratinocytes in interfollicular epidermis were con®ned to distinct columnar units representing epidermal proliferative units. The contribution of hair follicles to the epidermis was limited to a small rim of epidermis at the margin of the follicle, indicating that long term maintenance of interfollicular epidermis was independent of folliclederived cells. Our results indicate the presence of multiple stem cells in cutaneous epithelium, some with restricted lineages in the absence of major injury.

Epidermis is renewed by a population of stem cells that have been defined in vivo by slow turnover, label retention, position in the epidermis, and enrichment in ␤ 1 integrin, and in vitro by clonogenic growth, prolonged serial passage, and rapid adherence to extracellular matrix. 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 genetically labeled in culture by transduction with a retrovirus encoding the lacZ gene and grafted to athymic mice. Analysis of the cultures before grafting showed that 21.1-27.8% of clonogenic cells with the capacity for >30 generations were successfully transduced. In vivo, ␤-galactosidase (␤-gal) positive cells participated in the formation of a fully differentiated epithelium and were detected throughout the 40-week postgraft period, initially as loosely scattered clusters and later as distinct vertical columns. Viable cells recovered from excised grafts were seeded at clonal densities and 23.3-33.3% of the colonies thus formed were ␤-gal positive. In addition, no evidence of transgene inactivation was obtained: all keratinocyte colonies recovered from grafted tissue that were ␤-gal negative also lacked the lacZ transgene. These results show that cells with long-term growth properties in vitro do indeed persist in vivo and form a fully differentiated epidermis, thereby exhibiting the properties of stem cells.

Involucrin is a marker of keratinocyte terminal differentiation and is expressed only in the suprabasal layers of stratified squamous epithelium. In a previous study with various cell types in culture, we noted that expression of the putative human involucrin promoter was keratinocyte specific. To determine if this promoter is sufficient to direct expression to the suprabasal cells of stratified squamous epithelia in vivo, we have now generated transgenic mouse lines harboring the involucrin promoter sequences linked to a (3ga-lactosidase reporter gene. In the resulting lines, -galactosidase was expressed in the suprabasal compartment of stratified squamous epithelia and in hair follicles in a tissue-specific manner. In the palate, distinct vertical stacks of 3galacto-sidase-expressing cells were present, suggesting movement of clonally derived ceUs through the epithelium. The involucrin gene has a single intron upstream of the translational start site, and removal of this intron did not affect tissue-or stratumspecific expression. These results show that the 3.7-kb involucrin upstream sequences contain all the information necessary for a high level of tissue-and stratum-specific expression.

Renewal of epidermis is achieved by an ordered replication of stem cells and transit amplifying cells followed by terminal differentiation. In mouse epidermis, renewal is organized around highly ordered structures termed epidermal proliferative units (EPU), each generated by a single stem cell. It has been difficult to apply these concepts to the human epidermis where the basal layer is undulating and the strata have variable thickness. For example, it is unclear whether stem cells in human epidermis are located at the base of rete ridges or overlying the tip of dermal papilla. Data are available to support both views. To gain a better understanding of EPU organization in human skin, we have genetically marked xenografts of human foreskin with a lentivirus encoding a fluorescent marker protein and have mapped labeled columns of cells over a 28-wk period. By following these columns to their origin in the epidermis we have been able to determine that stem cells are dispersed along the basal compartment. The widths of these columns do vary considerably, with the narrowest originating from cells located in the base of the rete ridge. These findings provide new insights into the dynamics of epidermal renewal in human glabrous skin.