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 14-3-3σ 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.

Corneal renewal and repair are mediated by stem cells of the limbus, the narrow zone between the cornea and the bulbar conjunctiva. Ocular burns may destroy the limbus, causing limbal stem-cell deficiency. We investigated the long-term clinical results of cell therapy in patients with burn-related corneal destruction associated with limbal stem-cell deficiency, a highly disabling ocular disease.

We have analyzed the proliferative and differentiation potential of human ocular keratinocytes. Holoclones, meroclones, and paraclones, previously identified in skin, constitute also the proliferative compartment of the ocular epithelium. Ocular holoclones have the expected properties of stem cells, while transient amplifying cells have variable proliferative potential. Corneal stem cells are segregated in the limbus, while conjunctival stem cells are uniformly distributed in bulbar and forniceal conjunctiva. Conjunctival keratinocytes and goblet cells derive from a common bipotent progenitor. Goblet cells were found in cultures of transient amplifying cells, suggesting that commitment for goblet cell differentiation can occur late in the life of a single conjunctival clone. We found that conjunctival keratinocytes with high proliferative capacity give rise to goblet cells at least twice in their life and, more importantly, at rather precise times of their life history, namely at 45–50 cell doublings and at ∼15 cell doublings before senescence. Thus, the decision of conjunctival keratinocytes to differentiate into goblet cells appears to be dependent upon an intrinsic “cell doubling clock.” These data open new perspectives in the surgical treatment of severe defects of the anterior ocular surface with autologous cultured conjunctival epithelium.

Ocular burns cause depletion of limbal stem cells, which leads to corneal opacification and visual loss. Autologous cultured epithelial cells can restore damaged corneas, but this technology is still developing. We sought to establish a culture system that allows preservation of limbal stem cells and preparation of manageable epithelial sheets and to investigate whether such cultures can permanently restore total limbal stem cell deficiency. We selected a homogeneous group of patients whose limbal cell deficiency was evaluated by scoring the gravity of the clinical picture and the keratin expression pattern. Stem cells, obtained from the limbus of the contralateral eye, were cultivated onto a fibrin substrate and their preservation was evaluated by clonal analysis. Fibrin cultures were grafted onto damaged corneas. Fibrin-cultured limbal stem cells were successful in 14 of 18 patients. Re-epithelialization occurred within the first week. Inflammation and vascularization regressed within the first 3–4 weeks. By the first month, the corneal surface was covered by a transparent, normal-looking epithelium. At 12–27 months follow-up, corneal surfaces were clinically and cytologically stable. Three patients had a penetrating keratoplasty approximately 1 year after restoration of their corneal surface. Their visual acuity improved from light perception or counting fingers to 0.8–1.0. Preservation of limbal stem cells in culture gives new perspectives on the treatment of ocular disorders characterized by complete limbal stem cell deficiency. The multicenter nature of this study and the handiness and ease of long-distance transportation of the fibrin-cultured epithelial sheets suggest that this technology can now be widely applied.