Abstract
Purpose :
Hyaluronan (HA), an integral component of the limbal stem cell niche (LSCN), has been shown to be instrumental in maintaining limbal stem cells (LESCs) in vivo. Here, we investigated whether human LESCs also express HA in vitro. HA is known to assist LESCs in vivo, therefore, we also investigated whether this endogenous matrix supports ex vivoexpansion of LESCs and whether the addition of exogenous HA can further support LESCs.
Methods :
Primary human LESCs (hLESCs) were isolated from donor human corneas and a mouse corneal epithelial progenitor (TKE2) cell line was obtained. The HA extracellular matrix was identified in vitro through immunocytochemistry, flow cytometry, and a red blood exclusion assay. LESCs were cultured onto HA coated petri dishes or in the presence of HA through media supplementation. Cell viability, proliferation, cell size, colony formation capacity (CFC), and putative stem cell marker expression were investigated.
Results :
Both hLESCs and TKE2 cells present a HA rich ECM in vitro which is essential for maintaining viable LESCs. Providing exogenous HA as supplemented media increased LESC proliferation, CFC, and putative LESC marker expression.
Conclusions :
Exogenous and endogenous HA preserve the LESC phenotype supporting ex vivo expansion. The presence of HA in the ECM creates a specialized niche for LESCs both in vitro and ex vivo, therefore, supplementing hLESCs with HA can become an accessible and affordable option for clinical applications.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.