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Thore Schmedt, Yuming Chen, Shimin Li, Joseph A. Bonanno, Ula V. Jurkunas; Spontaneous And Telomerase Immortalization Of Primary Human Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1736.
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© ARVO (1962-2015); The Authors (2016-present)
Human corneal endothelium is composed of uniformly hexagonal cells whose main function is to maintain the clarity of the cornea by regulating stromal hydration through its pump and barrier functions. Although primary human corneal endothelial cells (HCEnCs) can be isolated from donor tissue, their proliferation rate is low, and the number of passages is very limited. Scarcity of donor tissue is a problem compounded by donor-to-donor variability. These major difficulties give rise to a substantial need for alternative in vitro model systems to study HCEnCs.
Primary HCEnCs were isolated from a 21-year-old male donor by EDTA treatment. Highly proliferative and regularly shaped HCEnCs (called HCEnC-21) were isolated at passage 9 and further passaged. A subset of HCEnC-21 cells was transduced with human telomerase reverse transcriptase (hTERT) cDNA using a retroviral vector (HCEnC-21T). The TRAP assay was employed to assess telomerase activity in protein extracts. Morphologic studies were done using phase-contrast microscopy. Gene expression was analyzed using Northern Blotting and TaqMan® real-time PCR. Biosynthesis and localization of proteins was confirmed by immunocytochemistry.
HCEnC-21 and HCEnC-21T exhibited increased proliferative capacity as compared to primary cells and maintained hexagonal morphology as well as monolayer formation and contact inhibition for over 40 passages. The cell doubling time was lower in HCEnC-21T than HCEnC-21 cells (p<0.01). HCEnC-21T expressed 78-fold higher hTERT mRNA levels (p<0.0001). HCEnC-21 and HCEnC-21T displayed 110-fold and 630-fold higher telomerase activity (p<0.05) compared to primary HCEnCs, respectively. ZO-1 localized to tight junctions in early (15) and late (30) passages of both, HCEnC-21 and HCEnC-21T cells, at confluence. Na/K ATPase A1, one of the major ion transporters, was produced and localized to the plasma membrane. HCEnC-21 and HCEnC-21T expressed critical ion transporters like Na/K ATPase A1, CA2, NHE1, MCT1, MCT2, AE2, CFTR and sAC10 at similar levels compared to primary HCEnCs and normal endothelium.
HCEnC-21 and HCEnC-21T maintain critical characteristics of normal human corneal endothelium without showing signs of cellular senescence and provide two powerful new tools for the in vitro study of corneal endothelial cell biology.
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