Abstract
Purpose :
Inflammation is a pivotal factor in the development of limbal stem cell deficiency. Limbal epithelial stem cells (LESCs) are both rare and essential for vision, thus ensuring their survival is imperative. Recent work from our lab establishes that the glycocalyx of LESCs is distinguished by a low abundance of the monosaccharide fucose. We hypothesize that low fucosylation of LESCs serves as a protective mechanism against cell death mediated by tumor necrosis factor-alpha (TNF-α), a cytokine implicated in limbal stem cell deficiency.
Methods :
A lectin-flow cytometry based sorting technique followed by a 10X Genomics single-cell RNA sequencing protocol was used to generate gene expression profiles of fucose-high and fucose-low cells from human limbal tissue. R’s Seurat package was used for differential gene analyses. Primary human limbal epithelial cells cultured with fucose inhibitor 2F-Peracetyl-Fucose (2FF) or vehicle control DMSO were treated with TNF-α and the death agonist 5Z-7-Oxozeaenol (5Z7). Cell death rate was measured via propidium iodide incorporation using live cell imaging and proliferation was assessed by analyzing population doubling metrics.
Results :
Over 90% of human LESCs, identified by high expression of canonical stem cell markers, had a low-fucose glycocalyx. Low-fucose LESCs had significantly reduced expression of pro-death genes FAS, BAX, BAK1, and BCL2L11 (BIM) and significantly increased expression of pro-survival gene BCL2 compared to high-fucose LESCs. Using ex-vivo primary human limbal cells, we found that inhibiting fucosylation significantly increased the resistance of these cells to TNF-α/5Z7-mediated cell death. Finally, fucose-inhibited primary limbal cells had greater cumulative population doublings and a significantly reduced population doubling time compared to vehicle control cells continuously exposed to TNF-α/5Z7.
Conclusions :
We identified a glycan-mediated process regulating limbal cell death. Our findings indicate fucose inhibition enhances survival of limbal epithelial cells challenged with inflammatory mediators. These data offer potential to enhance therapies for limbal stem cell deficiency and other inflammatory ocular diseases.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.