Abstract
Purpose :
ATP-binding cassette (ABC) superfamily member B5 (ABCB5) is a plasma membrane protein, which identifies limbal stem cells (LSCs) and can be used for prospective LSC isolation for therapeutic applications. Here we examined the diverse factors that influence the success of ABCB5-positive LSC recovery from human cadaveric donors.
Methods :
Whole eye globes (n=19 cases) preserved in moist chambers were purchased from the Saving Sight, Kansas City, MO and CorneaGen, Seattle, WA Eye Banks. The LSC isolation was performed within 1 week after death as follows: After removal of the central cornea and corneal endothelium, the remaining limbal tissue was incubated with Dispase II (MilliporeSigma, Burlington, MA) at 37°C for 1 hour. Subsequently, limbal epithelial cells were scraped and incubated with trypsin at 37°C for 30 minutes, stained with anti-ABCB5 mAb (clone 3C2) or the isotype-matched control mAb, and examined by flow cytometry using FACS Aria II (BD biosciences, San Jose, CA). The percentage of ABCB5-positive cells was determined by adjusting for the percentage of isotype control-positive cells using linear regression analyses. These results were correlated with the donors’ medical information provided by the Eye Banks. To identify the predictors of the ABCB5-positive LSC recovery, we performed stepwise regression analysis using the Akaike information criterion. All statistical analyses were performed with JMP® Pro 13.0.0 software (SAS Institute Inc., Cary, NC).
Results :
On average, 101.7±38.9 of ABCB5-positive LSCs per 10,000 dissociated limbal epithelial cells could be detected from 18 cases. No ABCB5-positive LSCs were detected in one case of a donor afflicted with scleroderma, which was excluded from the analyses. Increased age and cardiac disease-related death were associated with the lower yield of ABCB5-positive LSCs (P=0.05 and P=0.01, respectively). Intriguingly, the ABCB5-positive LSC yield was not affected by the time between death and LSC isolation (P=0.83). Additional stepwise regression analysis also identified cardiac disease-related death as the most important factor associated with the lower yield of ABCB5-positive LSCs (P=0.01).
Conclusions :
Our study suggests that age and cardiac disease-related death are factors that negatively influence the yield of LSCs. Within one week from the time of death, there was no significant correlation between the ABCB5-positive LSC yield and tissue processing time.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.