Abstract
Purpose: :
The corneal epithelium is suitable to distinguish keratinocyte stem and TA cells. Corneal stem cells are segregated in the limbus, limbal–derived TA cells form the corneal epithelium. The gene with the most striking effects on the development of squamous epithelia is p63. TP63 generates transactivating and N–terminus–truncated isoforms by alternative promoter usage. Alternative splicing gives rise to different C–termini designated alpha, beta, and gamma. DNp63alpha is the major isoform contained in human squamous epithelia, and its role is investigated.
Methods: :
Whole mount corneas and cultures obtained from different corneal areas were investigated by PCR, in situ hybridization,confocal microscopy immunofluorescence, western blot, colony forming efficiency, clonal analysis and counting total number of cell doublings. Mass cultures were transduced by lentiviral vectors and forced expression of transcription factors was induced in limbal cultures.
Results: :
We show that human limbal–corneal keratinocytes express not only DNp63alpha but also DNp63beta and gamma, which are contained in different compartments. This previously unrecognized expression pattern depends on the integrity of the epithelium. In resting corneas expression of DNp63alpha seems to be restricted to limbal stem cells and related to presence of another factor maintaining stem cells in the G0 phase of cell cycle. Instead, expression of DNp63beta and DNp63gamma correlates with limbal cell migration, corneal wound healing, corneal differentiation in wounded corneas and disappearance of this factor allowing the exit from G0 cell cycle phase. Limbal stem cells (holoclones) generate colonies expressing DNp63alpha, while corneal TA cells (paraclones) express DNp63beta and gamma. During corneal regeneration limbal p63+ cells endowed with a high proliferative capacity duplicate and migrate into the damaged cornea. We report clinical results obtained in a homogeneous group of 130 patients presenting chemical burn–dependent limbal stem cell deficiency, treated with cultured limbal cells.
Conclusions: :
DN alpha isoform of p63 seems to identify the stem cells whereas the proliferative or resting state of stem cells is modulated by the coexpression of other molecules.
Keywords: cornea: epithelium • transcription factors • gene/expression