June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Characterization of the Limbal Epithelial Stem Cell Niche
Author Affiliations & Notes
  • Isabel Yi Moreno
    College of Optometry, University of Houston, Houston, Texas, United States
  • Arian Parsaie
    College of Optometry, University of Houston, Houston, Texas, United States
    College of Natural Science and Mathematics, University of Houston, Houston, Texas, United States
  • Sudhir Verma
    College of Optometry, University of Houston, Houston, Texas, United States
    Deen Dayal Upadhyaya College, University of Delhi, New Delhi, Delhi, India
  • Tarsis Ferreira Gesteira
    College of Optometry, University of Houston, Houston, Texas, United States
  • Vivien Jane Coulson-Thomas
    College of Optometry, University of Houston, Houston, Texas, United States
  • Footnotes
    Commercial Relationships   Isabel Moreno None; Arian Parsaie None; Sudhir Verma None; Tarsis Gesteira None; Vivien Coulson-Thomas None
  • Footnotes
    Support  NIH/NEI Grant R01EY029289, PA-21-071
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 5150. doi:
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      Isabel Yi Moreno, Arian Parsaie, Sudhir Verma, Tarsis Ferreira Gesteira, Vivien Jane Coulson-Thomas; Characterization of the Limbal Epithelial Stem Cell Niche. Invest. Ophthalmol. Vis. Sci. 2023;64(8):5150.

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

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Abstract

Purpose : Limbal epithelial stem cells (LESCs) maintain the corneal epithelium during homeostasis and are necessary for regenerating the corneal epithelium after injury. LESCs have a specialized extracellular matrix, or niche, (LSCN) which helps maintain their stem cell properties and characteristics. We have recently identified an abundance of Hyaluronan (HA) present within the LSCN which is necessary for maintaining LESCs. In tissues, HA associates with various proteins and proteoglycans to form specific HA matrices with distinct physiological functions. Herein we characterize the HA specific matrix that is present within the LSCN.

Methods : Corneal buttons and limbal rims were dissected from wildtype C57BL6/J mice and subjected to mRNA extraction for sequencing using a NextSeq 500 (Illumina) sequencer. Data was processed using CLC Genomics Workbench 20 (Qiagen) and PPI Networks constructed using STRING database and visualized using Cytoscape with the MCODE plugin. Extracellular matrix related target genes were identified using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and their expression within the LSCN confirmed by real time PCR (qPCR), Western blotting (WB), and immunohistochemistry using mouse, pig, and human corneas. Prior to WB, samples were digested or not with hyaluronidase from Streptomyces (Millipore Sigma).

Results : Gene expression profiles were compared between the limbal region and the central cornea. 9,189 genes were found to be more highly expressed in the limbus, while 8,255 were more highly expressed in the central cornea, and 36 ECM related genes were found to be more highly expressed in the limbus, while 12 ECM related genes were more highly expressed in the central cornea. When analyzing the expression profile of hyaladherins, heavy chains (HCs) 2, 3, and 5, and tumor necrosis factor- stimulated gene-6 (TSG-6) were found to be more highly expressed in the limbal region, which was confirmed by real time PCR, WB and immunohistochemistry. HC2 and HC5 were the most highly expressed HCs identified. HA/HC-TSG-6 complexes were purified from the limbal region of mouse, human and porcine corneas and identified by WB.

Conclusions : HA/HC-TSG-6 complexes, similar to the complexes isolated from amniotic membranes for supporting LESCs, are expressed in the LSCN of mouse, porcine and human corneas. These HA/HC-TSG-6 complexes could be necessary for maintaining LESCs in vivo.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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