Abstract
Following a certain type-specific number of mitotic divisions, terminally differentiated cells undergo proliferative senescence, thwarting efforts to expand different cell populations in vitro for the needs of scientific research or medical therapies. The primary cause of this phenomenon is the progressive shortening of the telomeres and the subsequent activation of cell cycle control pathways leading to a block of cell proliferation. Restoration of telomere length by transgenic expression of telomerase reverse transcriptase (TERT) usually results in bypassing of the replicative senescence and ultimately in cell immortalization. To date, there have not been any reports regarding immortalization of cells from common marmoset (Callithrix jacchus), an important non-human primate model for various human diseases, with the use of exogenous human TERT (hTERT). In this study, marmoset fibroblasts were successfully immortalized with transposon-integrated transgenic hTERT and expanded in vitro for over 500 population doublings. Calculation of population doubling levels (PDL) showed that the derived hTERT-transgenic lines had significantly higher proliferation potential than the wild-type fibroblasts, which reached only a maximum of 46 doublings. However, the immortalized cells exhibited differences in the morphology compared with the control fibroblasts and transcriptome analysis also revealed changes in the gene expression patterns. Finally, the karyotypes of all hTERT-transgenic cell lines showed various aberrations such as presence of extra Chromosome 17, isochromosome 21q, or tetraploidy. By single-cell expansion of the least affected monoclonal immortalized line, one sub-clonal line with normal karyotype was established, suggesting the possibility to derive immortal marmoset cells with normal karyotypes. The results of this study are an important step towards the development and optimization of methods for the production of immortalized cells from common marmoset monkeys.
Highlights
The expansion of terminally differentiated somatic cells in vitro for research or medical therapy applications is limited by their finite proliferative lifespans
Exogenous human TERT (hTERT) prolongs cell proliferation potential in common marmoset fibroblasts To immortalize common marmoset fibroblasts, we used CAG promoter-driven hTERT (Fig 1A), which was inserted into the marmoset genome by piggyBac transposition
The hTERT-transgenic cells clearly showed higher proliferation potential compared with wild type primary fibroblasts (Fig 1B), as they underwent several hundred population doublings until the experiments were terminated
Summary
The expansion of terminally differentiated somatic cells in vitro for research or medical therapy applications is limited by their finite proliferative lifespans. The main reason for this restriction is the progressive shortening of telomere ends [2] leading to genomic DNA damage and activation of p53/p21-mediated cell cycle control pathways [3,4,5,6]. The cells may overcome this replication block due to defective or virus-suppressed p53 and Rb function [7, 8]; continued proliferation results in further shortening of telomeres, extensive chromosome damage, and genomic crisis leading to widespread apoptosis [5, 9]. Somatic cells express TR ubiquitously [11], but TERT is either absent or present at very low levels in senescent somatic cells [12] and is the prime determinant of telomerase activity. Immortalization with hTERT may have advantages over using tumor-inducing viruses or their components, such as Eppstein-Bar virus [29] or SV40 Large T antigen [30], which have been shown to cause malignancies and genomic aberrations
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