The proliferative compartment of stratified squamous epithelia consists of stem and transient amplifying (TA) keratinocytes. Some polypeptides are more abundant in putative epidermal stem cells than in TA cells, but no polypeptide confined to the stem cells has yet been identified. Here we show that the p63 transcription factor, a p53 homologue essential for regenerative proliferation in epithelial development, distinguishes human keratinocyte stem cells from their TA progeny. Within the cornea, nuclear p63 is expressed by the basal cells of the limbal epithelium, but not by TA cells covering the corneal surface. Human keratinocyte stem and TA cells when isolated in culture give rise to holoclones and paraclones, respectively. We show by clonal analysis that p63 is abundantly expressed by epidermal and limbal holoclones, but is undetectable in paraclones. TA keratinocytes, immediately after their withdrawal from the stem cell compartment (meroclones), have greatly reduced p63, even though they possess very appreciable proliferative capacity. Clonal evolution (i.e., generation of TA cells from precursor stem cells) is promoted by the sigma isoform of the 14-3-3 family of proteins. Keratinocytes whose 14-3-3 has been down-regulated remain in the stem cell compartment and maintain p63 during serial cultivation. The identification of p63 as a keratinocyte stem cell marker will be of practical importance for the clinical application of epithelial cultures in cell therapy as well as for studies on epithelial tumorigenesis.

R-spondins are a recently characterized small family of growth factors. Here we show that human R-spondin1 (RSPO1) is the gene disrupted in a recessive syndrome characterized by XX sex reversal, palmoplantar hyperkeratosis and predisposition to squamous cell carcinoma of the skin. Our data show, for the first time, that disruption of a single gene can lead to complete female-to-male sex reversal in the absence of the testis-determining gene, SRY.

In human epidermal keratinocytes, replicative senescence, is determined by a progressive decline of clonogenic and dividing cells. Its timing is controlled by clonal evolution, that is, by the continuous transition from stem cells to transient amplifying cells. We now report that downregulation of 14-3-3σ, which is specifically expressed in human stratified epithelia, prevents keratinocyte clonal evolution, thereby forcing keratinocytes into the stem cell compartment. This allows primary human keratinocytes to readily escape replicative senescence. 14-3-3σ–dependent bypass of senescence is accompanied by maintenance of telomerase activity and by downregulation of the p16INK4a tumor suppressor gene, hallmarks of keratinocyte immortalization. Taken together, these data therefore suggest that inhibition of a single endogenous gene product fosters immortalization of primary human epithelial cells without the need of exogenous oncogenes and/or oncoviruses.