June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Glycogene expression profile of human limbal epithelial cells with distinct clonogenic potential.
Author Affiliations & Notes
  • Pablo Argueso
    Department of Ophthalmology, Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Damien Guindolet
    Fondation Ophtalmologique A. de Rothschild, Paris, France
  • Ashley M. Woodward
    Department of Ophthalmology, Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Eric Gabison
    Fondation Ophtalmologique A. de Rothschild, Paris, France
  • Footnotes
    Commercial Relationships   Pablo Argueso None; Damien Guindolet None; Ashley Woodward None; Eric Gabison None
  • Footnotes
    Support  NIH Grant EY026147, Arthur Sachs Scholarship Fund.
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 1125. doi:
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      Pablo Argueso, Damien Guindolet, Ashley M. Woodward, Eric Gabison; Glycogene expression profile of human limbal epithelial cells with distinct clonogenic potential.. Invest. Ophthalmol. Vis. Sci. 2022;63(7):1125.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Glycans function as useful markers of stem cells but also regulate the ability of these cells to differentiate. Approximately 1–2% of the human genome encodes for genes involved in the biosynthesis of glycans. In the present study, we evaluated the expression of a small subset of glycogenes in human limbal epithelial cells with distinct clonogenic potential.

Methods : Human postmortem corneoscleral tissues were generously donated by the Lions VisionGift. Epithelial cells were cultured and expanded in vitro using growth-arrested mouse 3T3 fibroblasts. Individual clones were isolated with cloning cylinders and used in parallel for both colony formation and transcriptional assays. The analysis of 84 genes involved in the biosynthesis of glycans was carried out using a human RT2 Profiler PCR Array. Quantification of gene expression in primary cultures of human limbal epithelial cells was achieved by qPCR.

Results : Individual clones were classified as abortive or clonogenic based on the fraction of abortive colonies produced. Clones leading to >99% abortive colonies were referred as abortive while those with 50% or less as clonogenic. Analysis of glycogene expression in clonogenic colonies revealed a high content of transcripts regulating galactose and mannose metabolic pathways. Abortive colonies were characterized by increased levels of GCNT4 and FUCA2, two genes responsible for the branching of mucin-type O-glycans and the hydrolysis of fucose residues on N-glycans, respectively. Expansion of primary cultures of human limbal epithelial cells for 10 days resulted in stratification and a concomitant increase in MUC16, GCNT4 and FUCA2 expression.

Conclusions : These data indicate that the clonogenic potential of human limbal epithelial cells is associated with specific glycosylation pathways. Mucin-type O-glycan branching and increased fucose metabolism are linked to limbal epithelial cell differentiation.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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