Abstract
Purpose :
Hyaluronan (HA) is an essential limbal niche component that is required for maintaining limbal epithelial stem cells (LESCs). LESCs divide asymmetrically producing LESCs and transient amplifying cells (TACs) that migrate centripetally into the cornea. Studies have indicated that some TACs may present progenitor-like properties. Herein, we identified clusters of cells containing an HA-rich glycocalyx (HA clusters), in the peripheral cornea of both mice and humans. This study investigates whether these HA clusters in the peripheral cornea are associated with TACs with stem cell-like properties.
Methods :
We performed an integrated corneal whole mount analysis of the distribution of HA and LESCs progenies, using label retaining and lineage tracing models. With these models, HA clusters and TACs were tracked over 48 hours following a central corneal epithelium debridement wound. Specifically, we quantified the distribution and colocalization of TACs and HA across the limbal, peripheral, and central cornea using a semi-automatic script designed in Image J. The progenitor-like properties of cells within HA clusters were determined by isolating HA+ cells by magnetic-activated cell sorting (MACS), and subsequently analyzing cell size, colony formation capabilities, and staining for putative LESCs/TACs cell markers.
Results :
Based on all label retaining models used, there is a high co-localization rate between HA clusters and LRCs in the peripheral cornea of naïve wt mice, and wt mice 24 hours after a debridement wound. There is no longer a higher preponderance of LRCs within HA clusters in the peripheral cornea of wt mice 48 hours after a debridement wound. Mice deficient in HA, and therefore lacking HA clusters, present a significant loss of LRCs in the peripheral cornea. Only cells within HA clusters purified from the peripheral cornea present the ability to form holoclones, whereas cells outside HA clusters only form meroclones or paraclones.
Conclusions :
A subset of TACs within the peripheral cornea exist within clusters of cells that present an HA glycocalyx, coined HA clusters, and TACs within these HA clusters retain progenitor-like properties.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.