Abstract
Purpose :
Limbal epithelial stem cells (LSCs) are vital for maintaining corneal epithelium homeostasis and for resurfacing the cornea after injury. The LSC niche is composed of a specialized hyaluronan (HA) matrix that is necessary for maintaining viable LSCs. In the cornea, HA has been shown to be almost exclusively expressed in the limbal region. Our preliminary data show HA rich clusters also exist in the peripheral cornea. Herein we characterize these HA clusters.
Methods :
The distribution of LSCs, corneal epithelial progenitor cells (CEPCs), transient amplifying cells (TACs), and HA were analyzed in whole mounted corneas of wt (n=3) and HAS2Δ/ΔCorEpi mice (n=3) using label-retaining techniques. For label retaining, mice were pulsed with 5-ethynyl-2’-deoxyuridine (EdU) via daily intraperitoneal injections from P7-P14 and thereafter chased for 8-weeks. LRCs were detected using Click-iT™ EdU Alexa Fluor™ 488 Imaging Kit. 1.0 mm central debridement wounds were used to trigger the influx of TACs into the peripheral cornea 24 or 48 hours prior to euthanasia. Corneas were stained for HA using biotinylated HA binding protein and analyzed under the LSM 800 confocal microscope (Carl Zeiss Microscopy LLC).
Results :
After 8w chase, LRCs were mainly located in the limbal region, however, a small population LRCs were found within the corneal epithelium of all mice. HAS2Δ/ΔCorEpi mice, which have previously been shown to be LSC deficient, presented limited LRCs in the limbal region, and, significantly fewer LRCs in the cornea when compared to wt mice. Wt mice present a rich HA network throughout the limbal epithelium and HA clusters within the peripheral cornea. In contrast, HAS2Δ/ΔCorEpi mice presented limited HA expression within the limbal epithelium and few HA clusters within the peripheral cornea. A positive relationship was found between LRCs and HA clusters in naïve and wounded wt corneas 24h after wounding.
Conclusions :
A positive relationship was found between HA clusters and LRCs (including TACs), within the wt limbal and peripheral corneal epithelium in vivo. Thus, our preliminary data indicate TACs could exist in the peripheral cornea within HA clusters.
This is a 2021 ARVO Annual Meeting abstract.