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T. A. Fuchsluger, U. Jurkunas, A. Kazlauskas, R. Dana; Characteristics of Primary Human Corneal Endothelial Cells (pHCEC) and Immortalized HCEC (iHCEC) Studying Early Apoptosis and Gene Transfer. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1809.
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© ARVO (1962-2015); The Authors (2016-present)
The use of immortalized HCEC currently is widespread among cell death and gene transfer vision research investigators. This study evaluates characteristics of primary versus immortalized HCEC in early apoptosis and the response of these cells to gene transfer with empty vector (IZsGW, GFP) and the anti-apoptotic gene Bcl-xL.
Apoptosis was induced in immortalized and primary (passage 8) HCEC with an external (Actinomycin) and an internal (Etoposide) apoptotic inducer (0.1-100 micrograms/ml for 2-120h). Sensitivity towards Lentivirus and Adenovirus was examined using pHAGE-CMV-MCS-IZsGW (1.5 x 10^8 IU/ml) and AAV2-GFP (3.2 x 10^12 IU/ml, 3.2 x 10^3- 3.2 x 10^8; 24 hours of incubation, days 1-23). Flow cytometry was used to detect apoptosis with Annevin V-FITC and Propidium Iodide as well as expression rates of IZsGW and GFP. Apoptosis in Lenti-IZsGW-Bcl-xL versus Lenti-IZsGW (as empty vector, EV) was detected with Annexin V-PE, 7-AAD and flow cytometry.
Early Apoptosis rates peaked in iHCEC 6h and 30h after induction of apoptosis (up to 50% Ann+PI-), in pHCEC after 60h (up to 60% Ann+PI-). There was no significant dose dependency. In addition, pHCEC are much more sensitive to pHAGE-CMV-MCS-IZsGW than iHCEC, showing an IZsGreen expression rate of almost 100% already with 0.9x10^5 IU per 48well (iHCEC: max. 50% expression rate with 2.25x10^5 IU). The expression rates of AAV2-GFP reached a plateau of 80% in pHCEC 17 days after infection, in iHCEC after 23 days showing only 30% expression of GFP. Studying protection of HCEC with Lenti-IZsGr-BclxL resulted in early apoptosis in pHCEC of 63% (EV) being significantly reduced to 24% (see figures 1 and 2). In iHCEC there was no effect of EV versus BclxL resulting in 15-20% early apoptosis in each.
iHCEC deliver notably diverse data in apoptosis and gene transfer with lentivirus and adenovirus compared to pHCEC. Our data suggest that pHCEC provide more reliable data in studying apoptosis and gene transfer than iHCEC.
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