Abstract
Abstract: :
Purpose: To evaluate the correlation between cell size and gene expression of stem cell associated and differentiation markers by human corneal epithelial cells. Methods: Human corneal epithelial cells were cultured on a mitomycin C treated 3T3 fibroblast feeder layer using single cells isolated from fresh limbal tissues. Whole populations of confluent single cell cultures were sorted by a flow cytometer based on cell size. Four fractions of cells ranging in size from 8–12, 13–24, 25–36 and 37–48 µm in diameter were collected and subjected to total RNA extraction. Semiquantitative RT–PCR was performed for their expression of the stem cell associated markers: ΔNp63, ABCG2 and enolase–α, and the differentiation markers: K3, K12 and connexin 43. Results:Confluent primary human corneal epithelial cells had sizes ranging from 8–52 µm in diameter with 85–90% cells sized from 8 to 36 µm in diameter. The population with the smallest cell size (8–12 µm) sorted by flow cytometry accounted for 7.8 % of the whole population. These cells showed the highest levels of ΔNp63, ABCG2 and enolase–α mRNA but the lowest levels of K3, K12 and connexin 43 by semi–quantitative RT–PCR. There was an inverse correlation between the levels of ρNp63 and ABCG2 and cell size. The population with the biggest cell size (37–48 µm) showed low to barely detectable levels of ΔNp63, ABCG2 and enolase–α mRNA, but had the highest expression of K3 and K12. Conclusions:These findings suggest that the cell size correlates with molecular phenotype in human corneal epithelial cells. As cells enlarge, the expression of stem cell associated markers decreases while the levels of differentiation markers increases. Further studies are necessary to determine if the cell size correlates with cell proliferative capacity.
Keywords: cornea: epithelium • flow cytometry • gene/expression