Date Published: September 27, 2018
Publisher: Public Library of Science
Author(s): Stoyan Petkov, Tobias Kahland, Orr Shomroni, Thomas Lingner, Gabriela Salinas, Sigrid Fuchs, Katharina Debowski, Rüdiger Behr, Gabriele Saretzki.
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.
The expansion of terminally differentiated somatic cells in vitro for research or medical therapy applications is limited by their finite proliferative lifespans. Following a certain cell type-dependent number of mitotic divisions, most cells enter a phase of replicative senescence . The main reason for this restriction is the progressive shortening of telomere ends  leading to genomic DNA damage and activation of p53/p21-mediated cell cycle control pathways [3–6]. In some cases, the cells may overcome this replication block due to defective or virus-suppressed p53 and Rb function [7, 8]; however, continued proliferation results in further shortening of telomeres, extensive chromosome damage, and genomic crisis leading to widespread apoptosis [5, 9]. Restoration of the telomere length by telomerase, a ribonucleoprotein complex comprised of internal template RNA (TR) and a specific telomerase reverse transcriptase (TERT), leads to bypassing the crisis and, ultimately, to immortalization . Normally, somatic cells express TR ubiquitously , but TERT is either absent or present at very low levels in senescent somatic cells  and is therefore the prime determinant of telomerase activity. Various studies have demonstrated that overexpressing exogenous human TERT (hTERT) in the form of a transgene is sufficient to immortalize various cell types in the human [13–20] as well as in different animal species such as sheep , dog , pig , and rhesus macaque . While some groups have reported relatively normal phenotypes for hTERT-immortalized cells [16, 18–20], others have shown that such cells may exhibit cancer-associated changes and neoplastic transformation [25–28]. Nevertheless, immortalization with hTERT may have advantages over using tumor-inducing viruses or their components, such as Eppstein-Bar virus  or SV40 Large T antigen , which have been shown to cause malignancies and genomic aberrations.
The transposon-mediated transgenic hTERT expression in marmoset fibroblasts resulted in significant enhancement of the proliferation capacity and circumvented the Hayflick limit, as evidenced by PDL increase and longer proliferation lifespan of the immortalized cells. Compared to the control cells, which reached maximum of 45 PDL, the immortalized polyclonal line exceeded 500 PDL over the entire period of maintenance in culture without showing any signs of senescence. The sustained expression of hTERT in the transfected cell lines was confirmed by immunofluorescence as well as by RT-PCR. However, the immortalized fibroblast lines showed some marked differences compared with the control wild-type fibroblasts, such as reduced nuclear size and changes in cell morphology from typical mesenchymal to elongated, rather needle-like appearance (also at low densities). Transcriptome analysis suggested that these morphological changes were accompanied by a shift in the gene expression patterns as evidenced by the differential clustering of the immortalized cells separate from the non-immortalized control. Gene ontology analysis showed upregulation of genes related to various cellular processes, such as metabolism and cellular communication. While some genes were identified that could contribute to the changed cellular shape like the collagen encoding genes COL1A1 and COL1A2, no gene was found by transcriptome analysis that could directly be linked to the apparently reduced nuclear diameter.