Abstract
Purpose :
Fucosylation is a common carbohydrate modification found on many glycoproteins and glycolipids. It serves to modulate a vast array of biological functions such as tissue development, angiogenesis, cell adhesion and inflammation. In the present study, we evaluated the gene-expression profile of human limbal cells with different fucose profiles to better understand the epithelial stem cell microenvironment.
Methods :
Human cadaveric corneas were obtained from Lions VisionGift. Fluorescein-labeled Aleuria aurantia lectin (AAL), recognizing terminal fucose, was used for immunofluorescence and flow cytometry assays. Corneal epithelial cells were cultured and expanded in vitro using growth-arrested mouse 3T3 fibroblasts. AAL-sorted cells were used for colony-forming efficiency, population doubling and 10x single-cell RNA sequencing assays. Sequencing data were clustered using the Seurat R package.
Results :
Positive AAL staining was detected throughout the central corneal epithelium and in the apical portion of the limbus. By contrast, basal cells in the limbal epithelium, as well as in the hair follicle epithelium and the meibomian gland duct, were predominantly negative. Cell-sorting of limbal cells showing low AAL staining (AALlow) yielded increased colony formation and population doublings. Analysis by single-cell RNA sequencing revealed that the AALlow population consisted of melanocytes and immune cells whereas epithelial cells displayed different degrees of AAL staining. AALlow epithelial cells were enriched for stem cell markers including TP63, GPHA2, FRZB and CDH19. Further analysis of the AALlow population revealed that these cells had reduced expression of GDP-mannose-4,6-dehydratase, an enzyme catalyzing the first and regulatory steps in the de novo biosynthesis of GDP-fucose.
Conclusions :
The human limbus displays structural diversity in terms of glycan composition. Our data uncovers remarkable differences in cell surface fucosylation within epithelial cells and between different cell types in the limbal region and suggest important roles for this carbohydrate modification in maintaining the epithelial stem cell niche.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.