Differentially Expressed Genes Associated with Human Limbal Epithelial Phenotypes: New Molecules That Potentially Facilitate Selection of Stem Cell-Enriched Populations

Abstract

The aim of this study was to identify differentially expressed genes in the human limbal epithelium by microarray analysis. Total RNA isolates of human limbal and central corneal epithelia were used after transcription for hybridization on whole human genome expression microarrays. A set of differentially expressed genes detected by both microarrays was established. In the case of eight selected molecules, microarray results were confirmed by qRT-PCR, and protein expression in the cornea was examined by confocal immunofluorescence microscopy. Colocalization with the putative stem cell marker C/EBPδ was also examined. The authors established a database of 126 limbal overexpressed genes. qRT-PCR confirmed microarray results in all examined cases (SPON1, IFITM1, ITM2A, PHLDA1, CXCR4, FZD7, DCT, DKK4). Limbal localization of the protein product of SPON1, IFITM1, ITM2A, CXCR4, and DKK4 was shown with confocal immunofluorescence microscopy. SPON1, IFITM1, and ITM2A signals mostly colocalized with C/EBPδ-positive putative resting limbal stem cells. By detecting several new differentially expressed genes in the human corneal limbus, this study further expands current knowledge on the molecular signature of limbal epithelial stem cells. Plasma membrane localization of IFITM1 and ITM2A suggests their potential usefulness as targets to select stem cell-enriched populations from the limbal epithelium.