July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
ABCB5-positive limbal stem cell recovery from human donors
Author Affiliations & Notes
  • Yuzuru Sasamoto
    Medicine, Brigham and Women's Hospital, Boston, Massachusetts, United States
  • Naoko Sasamoto
    Obstetrics and Gynecology Epidemiology Center, Brigham and Women's Hospital, Boston, Massachusetts, United States
  • Bruce Ksander
    Schepens Eye Research Institute, Mass Eye & Ear, Massachusetts, United States
  • Markus Hermann Frank
    Transplant Research Program, Boston Children's Hospital, Massachusetts, United States
  • Natasha Frank
    Medicine, Brigham and Women's Hospital, Boston, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Yuzuru Sasamoto, Alcon Japan (F), Japan Eye Bank Association (F), Kanae Foundation for the Promotion of Medical Science (F); Naoko Sasamoto, None; Bruce Ksander, ABCB5-related US patent (P), NIH/NEI (F), Rheacell GmbH & Co. KG (P), Ticeba GmbH (P); Markus Frank, ABCB5-related US patent (P), NIH/NCI (F), NIH/NEI (F), Rheacell GmbH & Co. KG (P), Rheacell GmbH & Co. KG (C), Ticeba GmbH (P), Ticeba GmbH (C); Natasha Frank, NIH/NEI (F), Rheacell GmbH & Co. KG (P), Ticeba GmbH (P), VA (F)
  • Footnotes
    Support  NIH/NEI 1R24EY028767-01 grant to N.Y.F., B.R.K and M.H.F, NIH/NEI 1RO1EY025794-03 grant to N.Y.F., B.R.K and M.H.F, NIH/NCI grants R01CA113796, R01CA158467, and R01CA138231 to M.H.F., Department of Veterans Affairs VA Merit Review Awards VA BLR&D 1I01BX000516 and VA RR&D 1I01RX000989 to N.Y.F., the Kanae Foundation for the Promotion of Medical Science (Tokyo, Japan) to Y.S., Overseas grant from Japan Eye Bank Association (Tokyo, Japan) to Y.S, .
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 4136. doi:
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    • Get Citation

      Yuzuru Sasamoto, Naoko Sasamoto, Bruce Ksander, Markus Hermann Frank, Natasha Frank; ABCB5-positive limbal stem cell recovery from human donors. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4136.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

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