Yuzuru Sasamoto1, Naoko Sasamoto2, Johnathan Tran3, Ananda Mishra3, Bruce R Ksander4, Markus H Frank5, Natasha Y Frank6. 1. Division of Genetics, Brigham and Women's Hospital, Boston, MA, United States. 2. Obstetrics and Gynecology Epidemiology Center, Department of Obstetrics and Gynecology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, United States. 3. Division of Genetics, Brigham and Women's Hospital, Boston, MA, United States; Transplant Research Program, Boston Children's Hospital, Boston, MA, United States. 4. Massachusetts Eye & Ear Infirmary, Schepens Eye Research Institute, Boston, MA, United States. 5. Transplant Research Program, Boston Children's Hospital, Boston, MA, United States; Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA; Harvard Skin Disease Research Center, Department of Dermatology, Brigham and Women's Hospital, Boston, MA, USA; School of Medical and Health Sciences, Edith Cowan University, Perth, Western Australia, Australia. 6. Department of Medicine, VA Boston Healthcare System, Boston, MA, United States; Division of Genetics, Brigham and Women's Hospital, Boston, MA, United States; Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA. Electronic address: nyfrank@bwh.harvard.edu.
Abstract
PURPOSE: To identify factors associated with isolation yields of ATP-binding cassette (ABC) superfamily member B5 (ABCB5)-positive limbal stem cells (LSCs) from human cadaveric donor eyes. METHODS: Whole eye globes were obtained from the Saving Sight eye bank, Kansas City, MO and the CorneaGen eye bank, Seattle, WA. ABCB5-positive LSCs were sorted by flow cytometry upon anti-ABCB5 monoclonal antibody staining within one week after donor death. The yields of live limbal epithelial cells in their entirety and of isolated pure ABCB5-positive LSC subsets were correlated with variables contained in the eye donors' medical information. RESULTS: The mean isolation yield of live limbal epithelial cells and ABCB5-positive LSCs per donor eye was (340,000 ± 160,000 and 2,608 ± 1,842 respectively, mean ± SD). Stepwise regression analysis showed that cardiac disease-related death was the strongest negative predictor of the ABCB5-positive LSC isolation yield (p = 0.01). While we observed a trend for an age-related decline in the yield of ABCB5-positive LSCs, a statistically significant association could not be established (2% decrease/year, p = 0.11). Additionally, despite a trend for decreased isolation yields of total live limbal epithelial cells isolated from single donors with a longer time between death and tissue processing (p = 0.04), this did not affect the yields of purified ABCB5-positive LSC, which was independent of increasing time between death and tissue processing (p = 0.50). CONCLUSIONS: Our study identifies cardiac disease-related death as a donor variable significantly associated with lower ABCB5-positive LSC isolation yields.
PURPOSE: To identify factors associated with isolation yields of ATP-binding cassette (ABC) superfamily member B5 (ABCB5)-positive limbal stem cells (LSCs) from human cadaveric donor eyes. METHODS: Whole eye globes were obtained from the Saving Sight eye bank, Kansas City, MO and the CorneaGen eye bank, Seattle, WA. ABCB5-positive LSCs were sorted by flow cytometry upon anti-ABCB5 monoclonal antibody staining within one week after donordeath. The yields of live limbal epithelial cells in their entirety and of isolated pure ABCB5-positive LSC subsets were correlated with variables contained in the eye donors' medical information. RESULTS: The mean isolation yield of live limbal epithelial cells and ABCB5-positive LSCs per donor eye was (340,000 ± 160,000 and 2,608 ± 1,842 respectively, mean ± SD). Stepwise regression analysis showed that cardiac disease-related death was the strongest negative predictor of the ABCB5-positive LSC isolation yield (p = 0.01). While we observed a trend for an age-related decline in the yield of ABCB5-positive LSCs, a statistically significant association could not be established (2% decrease/year, p = 0.11). Additionally, despite a trend for decreased isolation yields of total live limbal epithelial cells isolated from single donors with a longer time between death and tissue processing (p = 0.04), this did not affect the yields of purified ABCB5-positive LSC, which was independent of increasing time between death and tissue processing (p = 0.50). CONCLUSIONS: Our study identifies cardiac disease-related death as a donor variable significantly associated with lower ABCB5-positive LSC isolation yields.
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