Abstract
Purpose :
Limbal epithelial stem cells (LESCs) reside within a LESC niche (LSCN). We recently identified that hyaluronan (HA) is a major constituent of the LSCN, and that HA is necessary for maintaining LESCs in the "stem cell" state, both in vitro and in vivo. Herein, we investigated whether there is a loss of the HA-rich matrix within the LSCN of human donor corneas over time in storage and if so, does it correlate with a loss of LESCs.
Methods :
Human donor corneas preserved in optisol-GS storage media, ranging from 1 to 12 days, were processed for histology and corneal and limbal epithelium morphology was assessed with H&E staining. Hyaluronan expression was evaluated by immunohistochemistry using biotinylated HA binding protein, and HA synthase (HAS) expression was quantified by real time PCR (qPCR) in the corneal and limbal epithelium. LESCs were identified by both immunocytochemistry and qPCR against K15, K8, K19, DNp63, ABCB5 and ABCG2. The integrity of the HA-rich LSCN was correlated with the number of LESCs present in the limbal region.
Results :
There is a loss of HA within the LSCN of human donor corneas with time in storage media. A rich HA matrix is present within the limbal region of corneas within 2 days of storage however, there is a significant loss of HA beyond 3 days in storage media. Both Has1 and Has2 are expressed in the limbal region of human donor corneas, and there is a ~ 6-fold decrease in Has 2 between corneas that were stored for 7-10 days compared to the ones stored for 2-3 days. No significant changes in Has1 expression were observed with time in storage. The loss of HA within the LSCN correlates with a loss of LESCs and a loss of epithelial integrity. Corneas presenting low levels of HA within the LSCN correlate with the epithelium sloughing off during storage.
Conclusions :
The composition of HA-rich LSCN of human donor corneas changes over time during hypothermic storage, with a significant loss of HA over time. The loss of HA correlates with a loss in the number of viable LESCs.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.