Introduction of the hTERT Vadimezan site catalytic subunit [15]. Similarly, different types of mitotically competent somatic cells such as epithelial cells [16,17] and vascular endothelial cells [18] have been found to be responsive to hTERT expression, leading to the development of proliferative and phenotypically specific cell lines. In contrast, immortalization with hTERT alone has been problematic in mitotically incompetent somatic cells such as neural, glial, and muscle cells, thus immortalization with hTERT has only been reported for their replicating progenitors, not for the terminally differentiated cells themselves [19]. HCEn is another example of terminally differentiated somatic cells that, like neuronal tissue, 23727046 are neural crest-derived and mitotically arrested. Moreover, there is a growing need to generate corneal endothelial cell lines to study disease processes, especially premature depletion of cells in vivo, resulting in corneal blindness. The development of reliable and long-lasting cell culture systems is of eminent importance to provide better models for the study of HCEnC biology and regeneration. In this manuscript, we investigated whether hTERT expression alone is sufficient to immortalize human corneal endothelial cells. We detected that primary endothelial cell cultures exhibit distinct subpopulations of endothelial cells that, after isolation, were conducive to hTERT immortalization. A highly uniform subpopulation of endothelial cells (HCEnC-21) was derived from primary cells harvested from a 21-year-old male (21M) donor. Following transduction with hTERT, HCEnC-21 yielded highly hTERT-expressing cells (HCEnC-21T). To our knowledge, this is the first report of a corneal endothelial cell immortalization that is not based on oncogene expression, and that is able to simultaneously preserve high proliferative activity, as well as corneal endothelial morphology, marker characteristics, and functionality.that retain corneal endothelial characteristics, the morphological differences among cells in a series of primary cultures from different donor corneas were investigated. Among largely nonproliferative and senescent primary cells from 21M, a subpopulation of cells growing in colony-like structures was detected (Figure 1D). These colonies consisted of regularly shaped hexagonal cells that did not exhibit JRF 12 web fibroblast-like morphology and were significantly smaller than the rest of 21M primary cells. Selective isolation of morphologically distinct colonies was performed, and the cells were continuously passaged, avoiding contamination with senescent cells by monitoring cellular morphology. These cells were designated HCEnC-21. In addition, identification, isolation, and continued passaging of a phenotypically distinct population of cells with highly uniform polygonal morphology were performed in primary cultures from 56- and 70year-old donor corneas (Figure S1).Telomerase Increases Corneal Endothelial Proliferative Capacity without Loss of P53 FunctionTo investigate the role of hTERT overexpression on corneal endothelial cell proliferation, 21M primary and HCEnC-21 cells were transduced with hTERT mRNA (21M+hTERT and HCEnC-21T, respectively). Successful transduction was indicated by significantly increased hTERT mRNA levels in 21M+hTERT (979-fold, P = 0.00019) and HCEnC-21T (373-fold, P = 0.000017) cells (Figure 2A). Notably, HCEnC-21 cells expressed 5-fold (P = 0.00011) more hTERT mRNA than non-transduced 21M primary cells. However, des.Introduction of the hTERT catalytic subunit [15]. Similarly, different types of mitotically competent somatic cells such as epithelial cells [16,17] and vascular endothelial cells [18] have been found to be responsive to hTERT expression, leading to the development of proliferative and phenotypically specific cell lines. In contrast, immortalization with hTERT alone has been problematic in mitotically incompetent somatic cells such as neural, glial, and muscle cells, thus immortalization with hTERT has only been reported for their replicating progenitors, not for the terminally differentiated cells themselves [19]. HCEn is another example of terminally differentiated somatic cells that, like neuronal tissue, 23727046 are neural crest-derived and mitotically arrested. Moreover, there is a growing need to generate corneal endothelial cell lines to study disease processes, especially premature depletion of cells in vivo, resulting in corneal blindness. The development of reliable and long-lasting cell culture systems is of eminent importance to provide better models for the study of HCEnC biology and regeneration. In this manuscript, we investigated whether hTERT expression alone is sufficient to immortalize human corneal endothelial cells. We detected that primary endothelial cell cultures exhibit distinct subpopulations of endothelial cells that, after isolation, were conducive to hTERT immortalization. A highly uniform subpopulation of endothelial cells (HCEnC-21) was derived from primary cells harvested from a 21-year-old male (21M) donor. Following transduction with hTERT, HCEnC-21 yielded highly hTERT-expressing cells (HCEnC-21T). To our knowledge, this is the first report of a corneal endothelial cell immortalization that is not based on oncogene expression, and that is able to simultaneously preserve high proliferative activity, as well as corneal endothelial morphology, marker characteristics, and functionality.that retain corneal endothelial characteristics, the morphological differences among cells in a series of primary cultures from different donor corneas were investigated. Among largely nonproliferative and senescent primary cells from 21M, a subpopulation of cells growing in colony-like structures was detected (Figure 1D). These colonies consisted of regularly shaped hexagonal cells that did not exhibit fibroblast-like morphology and were significantly smaller than the rest of 21M primary cells. Selective isolation of morphologically distinct colonies was performed, and the cells were continuously passaged, avoiding contamination with senescent cells by monitoring cellular morphology. These cells were designated HCEnC-21. In addition, identification, isolation, and continued passaging of a phenotypically distinct population of cells with highly uniform polygonal morphology were performed in primary cultures from 56- and 70year-old donor corneas (Figure S1).Telomerase Increases Corneal Endothelial Proliferative Capacity without Loss of P53 FunctionTo investigate the role of hTERT overexpression on corneal endothelial cell proliferation, 21M primary and HCEnC-21 cells were transduced with hTERT mRNA (21M+hTERT and HCEnC-21T, respectively). Successful transduction was indicated by significantly increased hTERT mRNA levels in 21M+hTERT (979-fold, P = 0.00019) and HCEnC-21T (373-fold, P = 0.000017) cells (Figure 2A). Notably, HCEnC-21 cells expressed 5-fold (P = 0.00011) more hTERT mRNA than non-transduced 21M primary cells. However, des.
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