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Sajjad Ahmad, Arianna Bianchi, Dean Hallam, Gabriele Saretzki; Limbal Stem Cells, Telomerase Activity And Telomere Length Decline With Increasing Culture Of Human Limbal Epithelium. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5134.
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© ARVO (1962-2015); The Authors (2016-present)
Corneal epithelial stem cells, or limbal stem cells as they are commonly known, reside in the limbal epithelium. Limbal stem cells can be expanded by culturing human limbal epithelium for research and clinical applications. Telomeres protect the ends of chromosomes and decrease in length during ageing. Telomerase is an enzyme which elongates telomeres thereby preventing cellular ageing. The purpose of this study was to determine telomere length and telomerase activity in human limbal epithelial cultures from donors of different ages and with increasing culture passage.
Human limbal tissue, donated for research, was obtained from donors of varying ages. Limbal epithelial cultures were established from these tissues by co-culture with inactivated 3T3-J2 mouse fibroblasts. Once established, cultures were serially passaged up to passage 3. At each passage, cultured limbal epithelial cells were analysed for: colony forming efficiency, real time PCR for putative limbal stem cell markers p63 and ABCG2, TRAP ELISA assay for telomerase activity, and real time PCR for telomere length.
Limbal epithelial cultures were established from donors of varying ages (61-85 years). Colony forming efficiency and p63 and ABCG2 expression declined with increasing passage in all the cultures (p<0.01). During passage 1 colony forming efficiency was noted to decline with increasing donor age with statistical significance. Telomerase activity and telomere length also declined with increasing passage but were not found to be correlated with donor age.
The relative amount of limbal stem cells declines with increasing passage as determined by colony forming efficiencies and the real time PCR data for putative limbal stem cell markers. Interestingly, colony forming efficiency during passage 1 correlates significantly with donor age. Telomerase activity and telomere length also decline with increasing passage number indicating an increase in cellular ageing. Whether these indicators of cellular ageing are related to a decline in the limbal stem cells present within the cultures or whether it is related to increasing oxidative stress in culture remains to be determined, and further studies are underway to address this.
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