1,2Aboalchamat B., 2Wallis C., 1Engelmann K., 2Allen M., 2Faragher R.

1Universitäts-Augenklinik Eppendorf, Hamburg Germany; 2School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton BN2 4GJ, UK

Purpose: To investigate the mechanisms controlling replicative lifespan and differentiation status in cultured human corneal endothelial cells (HCEC).
Methods: Expanded cultures of HCEC were infected with a retroviral vector containing the catalytic subunit of human telomerase (hTERT). Telomere lengths were determined both pre and post infection by TRF analysis and the presence of telomerase activity using the TRAP assay. Human keratocytes (EK1.BR) were used as a control. HCEC were grown in culture and the presence of an endothelial-specific signature channel was also determined by whole cell patch clamp analysis.
Results: Ectoptic expression of hTERT restored telomerase activity and prolonged telomeres in both Ek1.Br and HCEC. However whilst human keratocytes (EK1.BR) immortalised in the presence of telomerase there was no effect on the replicative lifespan of human corneal endothelial cells. Isolated HCEC retained the temperature sensitive and anion activated potassium channel independent from the morphological appearance and proliferative age.
Conclusion: These data indicate that cultured HCEC retain the distinguishing electrophysiological characteristics of intact corneal endothelium and that the cell type possesses a telomere-independent divisional counting system. This is the first ocular cell type for which this has been shown to be true.
*Dr. Bilal Aboalchamat was supported by the DFG (AB 120/2-1).

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