Despite the central role of human epidermal stem cells in tissue homeostasis, wound repair, and neoplasia, remarkably little is known about these cells, largely due to the absence of molecular markers that distinguish them from other proliferative cells within the germinative͞basal layer. Epidermal stem cells can be distinguished from other cells in the basal layer by their quiescent nature in vivo and their greater overall proliferative capacity. In this study, we demonstrate enrichment and isolation of a subpopulation of basal epidermal cells from neonatal human foreskin based on cell surface phenotype, which satisfy these criteria. These putative stem cells are distinguished from other basal cells by their characteristic expression of high levels of the adhesion molecule ␣ 6 , a member of the integrin family (␣ 6 bri ), and low levels of a proliferation-associated cell surface marker recognized by recently described mAb 10G7 (10G7 dim ). We conclude that cells with the phenotype ␣ 6 bri 10G7 dim represent the epidermal stem cell population based on the demonstration that these cells (i) exhibit the greatest regenerative capacity of any basal cells, (ii) represent a minor subpopulation (Ϸ10%) of immature epidermal cells, which (iii) are quiescent at the time of isolation from the epidermis, as determined by cell cycle analysis.

Abstract. We have examined cultures of neonatal human foreskin keratinocytes (HFKs) to determine the ligands and functions of integrins 0~2/31, and ot3/31 in normal epidermal stratification and adhesion to the basement membrane zone (BMZ) in skin. We used three assay systems, HFK adhesion to purified extracellular matrix (ECM) ligands and endogenous secreted ECM, localization of integrins in focal adhesions (FAs), and inhibition of HFK adhesion with mAbs to conclude: (a) A new anti-ot3fll mAb, P1F2, localized ot3/~l in FAs on purified laminin > fibronectin/collagen, indicating that laminin was the best exogenous ligand for ct3~l. However, in long term culture, ot3/~l preferentially codistributed in and around FAs with secreted laminin-containing ECM, in preference to exogenous laminin. Anti-a3/~l, mAb P1B5, detached prolonged cultures of HFKs from culture plates or from partially purified HFK ECM indicating that interaction of a3/~l with the secreted laminincontaining ECM was primarily responsible for HFK adhesion in long term culture. (b), In FA assays, a2/~l localized in FAs coincident with initial HFK adhesion to exogenous collagen, but not laminin or fibronectin. However, in inhibition assays, anti-&2/31 inhibited initial HFK adhesion to both laminin and collagen. Thus, ot2/31 contributes to initial HFK adhesion to laminin but tx3/31 is primarily responsible for longterm HFK adhesion to secreted laminin-containing ECM. (c) Serum or Ca2+-induced aggregation of HFKs resulted in relocation of 0t2/31 and ot3/31 from FAs to cell-cell contacts. Further, cell-cell adhesion was inhibited by anti-c~3/31 (P1B5) and a new anti-/31 mAb (P4C10). Thus, interaction of c~3/31 with either ECM or membrane coreceptors at cell-cell contacts may facilitate Ca2+-induced HFK aggregation. (d) It is suggested that interaction of ot3/31 with a secreted, laminin-containing ECM in cultured HFKs, duplicates the role of ot3/31 in basal cell adhesion to the BMZ in skin. Further, relocation of o~2B1 and c~3/31 to cell-cell contacts may result in detachment of cells from the BMZ and increased cell-cell adhesion in the suprabasal cells contributing to stratification of the skin.

The Drosophila cuticle is essential for maintaining the surface barrier defenses of the fly. Integral to cuticle resilience is the transcription factor grainy head, which regulates production of the enzyme required for covalent cross-linking of the cuticular structural components. We report that formation and maintenance of the epidermal barrier in mice are dependent on a mammalian homolog of grainy head, Grainy head-like 3. Mice lacking this factor display defective skin barrier function and deficient wound repair, accompanied by reduced expression of transglutaminase 1, the key enzyme involved in cross-linking the structural components of the superficial epidermis. These findings suggest that the functional mechanisms involving protein cross-linking that maintain the epidermal barrier and induce tissue repair are conserved across 700 million years of evolution.