Pite high hTERT expression, 21M+hTERT cells were slow growing and developed typical signs of cellular senescence at the same passages as non-transduced cells (Figure 1I). Similarly, hTERT transduction of primary cells from the 56- and 70-year-old donors did not result in an extended life span. In contrast, both HCEnC-21 and HCEnC-21T cells have been proliferating for more than 70 passages (in 15 mo) without signs of senescence. Importantly, phase-contrast microscopy revealed that HCEnC-21 (Figures 1E and S2) and HCEnC-21T (Figures 1G and S2) continuously grew in monolayers in a contact-inhibited fashion and maintained their endothelial hexagonal MNS web morphology at all passages. To confirm the functionality of hTERT detected in HCEnC-21 and HCEnC-21T, telomerase enzyme get LY-2409021 activity in cell lysates was assayed using the telomeric repeat amplification protocol (TRAP) (Figure 2B). Heat-inactivated cell extracts served as an internal negative control, since hTERT is a temperature-sensitive enzyme. While the telomerase activity of 21M primary cells was not significantly different from the respective heat-inactivated control, indicating the absence of telomerase activity in 21M, HCEnC-21T cells displayed a significant increase in telomerase activity of about 505-fold (P = 0.021) relative to 21M as deduced from the template copy numbers. Importantly, HCEnC-21 cell extracts exhibited a 110-fold (P = 0.00062) increase in telomerase activity compared to 21M, confirming the presence of endogenous telomerase activity in HCEnC-21. To explore differences in the proliferative capacity, the cell doubling times (CDTs) of HCEnC-21 and HCEnC-21T cells were determined for earlier (19?4) and later (32?1) passages (Figure 2C). HCEnC-21 and HCEnC-21T cells had similar CDTs of around 30 hr at earlier passages. At later passages, HCEnC21T cells maintained a high proliferation rate showing no significant difference in CDT, whereas HCEnC-21 cells exhibited a significantly increased CDT (39 hr, P = 0.04), indicating slower cell proliferation. This suggests that higher hTERT activity leads to a higher proliferation rate of HCEnC-21T cells compared to HCEnC-21 in long-term cell cultures.Results Corneal Endothelial Primary Cell Cultures Consist of Different Subpopulations of CellsThe HCEn is a monolayer of 1317923 highly uniform cells with characteristic hexagonal morphology that are non-proliferative in vivo (Figure 1A). HCEnCs can be isolated and cultured in vitro; however, their proliferative potential is very low, and primary HCEnCs show a strong tendency to enter senescence. Figure 1B shows a phase-contrast micrograph of passage-9 primary HCEnCs. These were isolated from a 21-year-old donor (21M) and displayed typical signs of senescence, e.g. large and flat morphology, binucleation, and granulation. In addition, even early-passage primary HCEnCs are highly prone to EMT, which changes the cell phenotype into an elongated and fibroblast-like morphology. Figure 1C depicts primary HCEnCs isolated from a 63-year-old donor that underwent EMT at passage 1 resulting in the loss of corneal endothelial-specific hexagonal morphology and transformation into an elongated and fibroblast-like abnormal phenotype. To establish corneal endothelial long-term cell culturesTelomerase-Immortalized Human Corneal EndotheliumFigure 1. Morphologic study of corneal endothelial primary cells. (A) In vivo confocal microscopy of human corneal endothelium demonstrating the typical hexagonal cell morphology.Pite high hTERT expression, 21M+hTERT cells were slow growing and developed typical signs of cellular senescence at the same passages as non-transduced cells (Figure 1I). Similarly, hTERT transduction of primary cells from the 56- and 70-year-old donors did not result in an extended life span. In contrast, both HCEnC-21 and HCEnC-21T cells have been proliferating for more than 70 passages (in 15 mo) without signs of senescence. Importantly, phase-contrast microscopy revealed that HCEnC-21 (Figures 1E and S2) and HCEnC-21T (Figures 1G and S2) continuously grew in monolayers in a contact-inhibited fashion and maintained their endothelial hexagonal morphology at all passages. To confirm the functionality of hTERT detected in HCEnC-21 and HCEnC-21T, telomerase enzyme activity in cell lysates was assayed using the telomeric repeat amplification protocol (TRAP) (Figure 2B). Heat-inactivated cell extracts served as an internal negative control, since hTERT is a temperature-sensitive enzyme. While the telomerase activity of 21M primary cells was not significantly different from the respective heat-inactivated control, indicating the absence of telomerase activity in 21M, HCEnC-21T cells displayed a significant increase in telomerase activity of about 505-fold (P = 0.021) relative to 21M as deduced from the template copy numbers. Importantly, HCEnC-21 cell extracts exhibited a 110-fold (P = 0.00062) increase in telomerase activity compared to 21M, confirming the presence of endogenous telomerase activity in HCEnC-21. To explore differences in the proliferative capacity, the cell doubling times (CDTs) of HCEnC-21 and HCEnC-21T cells were determined for earlier (19?4) and later (32?1) passages (Figure 2C). HCEnC-21 and HCEnC-21T cells had similar CDTs of around 30 hr at earlier passages. At later passages, HCEnC21T cells maintained a high proliferation rate showing no significant difference in CDT, whereas HCEnC-21 cells exhibited a significantly increased CDT (39 hr, P = 0.04), indicating slower cell proliferation. This suggests that higher hTERT activity leads to a higher proliferation rate of HCEnC-21T cells compared to HCEnC-21 in long-term cell cultures.Results Corneal Endothelial Primary Cell Cultures Consist of Different Subpopulations of CellsThe HCEn is a monolayer of 1317923 highly uniform cells with characteristic hexagonal morphology that are non-proliferative in vivo (Figure 1A). HCEnCs can be isolated and cultured in vitro; however, their proliferative potential is very low, and primary HCEnCs show a strong tendency to enter senescence. Figure 1B shows a phase-contrast micrograph of passage-9 primary HCEnCs. These were isolated from a 21-year-old donor (21M) and displayed typical signs of senescence, e.g. large and flat morphology, binucleation, and granulation. In addition, even early-passage primary HCEnCs are highly prone to EMT, which changes the cell phenotype into an elongated and fibroblast-like morphology. Figure 1C depicts primary HCEnCs isolated from a 63-year-old donor that underwent EMT at passage 1 resulting in the loss of corneal endothelial-specific hexagonal morphology and transformation into an elongated and fibroblast-like abnormal phenotype. To establish corneal endothelial long-term cell culturesTelomerase-Immortalized Human Corneal EndotheliumFigure 1. Morphologic study of corneal endothelial primary cells. (A) In vivo confocal microscopy of human corneal endothelium demonstrating the typical hexagonal cell morphology.
