Abstract
Purpose :
Previously we have shown the regulatory roles of limbal stromal cell (LSC)-derived exosomes (Exos) in normal (N) and diabetic (DM) limbal epithelial cells (LEC) in vitro and ex vivo organ-cultured corneas. Our purpose was to evaluate the effects of N and DM LEC-derived Exos in N and DM LSC maintenance, proliferation and wound healing.
Methods :
Human autopsy age-matched N and DM eyes were from the National Disease Research Interchange. LEC and LSC were isolated from dissected corneas using Dispase/Trypsin and collagenase type IV, respectively. Exosomes were prepared from N and DM LEC-conditioned media by ultracentrifugation. LEC-derived Exos were characterized by NanoSight for morphology and particle size, and by flow cytometry and western blot for their markers (CD63, CD81). Exos were labeled with PKH-67 dye to determine their uptake by LSCs and LECs using confocal microscopy. MTS proliferation assay and scratch wound assay were performed on Exo-treated LSC. Markers of mesenchymal stem cells (MSC) and differentiated keratocytes were assessed by western blot.
Results :
Isolated Exos sizes ranged between 50-200 nm with the typical cup shape morphology. N and DM Exos were positive for CD63 and CD81 by both western blot and flow cytometry. The uptaken Exos were observed by confocal microscopy and confirmed by FACS analysis in PKH-67-labeled Exo treated LSC and LECs. FACS analysis showed significant five-fold higher LEC-derived Exo uptake by LSCs than LECs. N Exo treatment significantly stimulated LSC wound healing and proliferation, with the stronger effect than with DM Exo treatment. N Exo treatment increased, whereas DM Exo decreased the protein level of MSC marker, CD90, in N LSCs compared to untreated control by western blot. On the contrary, treatment of N LSCs with N Exo decreased keratocyte markers, ALDH3A1, lumican and keratocan, with a less pronounced effect after DM Exo treatment.
Conclusions :
The increased uptake of LEC-derived Exos by LSCs than by LECs suggests the cross-talk between limbal epithelial and stromal cells. The greater effects of N Exos than DM Exos on cell proliferation, wound healing and marker expressions may be due to the differences in their cargos.
This is a 2021 ARVO Annual Meeting abstract.