Expression of p63 in Cultured Corneal Epithelium is a Function of Cell Confluence

Abstract

Abstract: : Purpose: Recently there has been much controversy over whether p63, a homologue of the transcription factor p53, is exclusively expressed in corneal epithelial stem cells. We investigated p63 expression pattern in cultured corneal epithelial cells and its relation to cell confluence and expression of corneal lineage specific differentiation marker keratin 3 (K3). Methods: Passage (p) 2–4 cultured limbal corneal epithelial cells were plated at either 2.5 (sparse) or 5 (dense) x 10⁴ cells per cm². Cultures were fixed and evaluated at day 1 and day 4. Phase contrast and fluorescent microscopic images were overlapped to correlate cell morphology and the stage of cell confluence with antigen expression patterns. Representative views of 3 different slides were independently scored by 2 investigators for percent cells expressing p63 and K3. Results were compared by one–way ANOVA. Results: More than 85% (85– 90%) of cells expressed p63 at Day 1 regardless of cell plating density (check student t–test). Densely plated cells were confluent at day 4 and demonstrated markedly reduced p63 expression, with only 16.9% positive (p=0.001 compared to Day 1 dense cultures). In contrast, sparsely plated cells were subconfluent on Day 4 and demonstrated high levels of p63 expression (87.4%, p= 0.02 compared to Day 4 dense cultures). Morphologic observations in the confluent cultured dishes included an increase in cell–cell contact, a decrease in the number of mitotic cells, and a large number of cells with low nuclear to cytoplasmic ratio. K3 expression was lower in the sparsely plated subconfluent cultures as compared to their confluent counterparts. Conclusions: Most (about 90%) corneal epithelial cells express p63 in the first 24 hours of cell culture. These p63+ cells most likely represent corneal epithelial stem cells as well as transiently amplifying (TA) cells. While cultures remain sub–confluent, the expression of p63 does not change over time. Cell–cell contact seems to be the main signal for halting cell division, decreasing p63 expression, and inducing differentiation in confluent cultures. The decreased number of remaining p63+ cells in the confluent cultures may represent potential stem cells. It is possible that during times of rapid cell turnover, such as in subconfluent culture conditions, p63 is expressed by stem cells as well as TA cells, while in quiescent confluent cultures, p63 expression is more restricted to the corneal epithelial stem cells.

Keywords: cornea: basic science • cornea: epithelium • microscopy: light/fluorescence/immunohistochemistry