Gene Expression Profile in Immortalized Human Periodontal Ligament Fibroblasts Through hTERT Ectopic Expression: Transcriptome and Bioinformatic Analysis

Abstract

Human periodontal ligament fibroblast (hPLF) cells play an important role in maintaining oral cavity homeostasis with special function in tissue regeneration and maintenance of dental alveoli. Although their primary cell cultures are considered a good experimental model with no genetic changes, the finite life span may limit some experimental designs. The immortalization process increases cell life span but may cause genetic changes and chromosomal instability, resulting in direct effects on physiological cell responses. In this way, we aimed to investigate the global gene expression of hPLFs after the immortalization process by the ectopic expression of the catalytic subunit of the enzyme telomerase reverse transcriptase (hTERT) through transcriptome analysis. The embryonic origin of the primary culture of hPLF cells and immortalized hPLF-hTERT was also tested by vimentin staining, hTERT synthesis evaluated by indirect immunocytochemistry, analysis of cell proliferation, and morphology. The results indicated that hPLFs and hPLF-hTERT were positive for vimentin. On the 20th cell passage, hPLFs were in senescence, while hPLF-hTERT maintained their proliferation and morphology characteristics. At the same passage, hPLF-hTERT presented a significant increase in hTERT synthesis, but transcriptome did not reveal overexpression of the hTERT gene. Fifty-eight genes had their expression altered (11 upregulated and 47 downregulated) with the absence of changes in the key genes related to these cell types and in the main cancer-associated genes. In addition, the increase in hTERT protein expression without the overexpression of its gene indicates posttranscriptional level regulation. Successful immortalization of hPLFs through the ectopic expression of hTERT encourages further studies to design experimental protocols to investigate clinical questions from a translational perspective.

Characteristics of Whale Müller Glia in Primary and Immortalized Cultures

“…The number of passages before reaching senescence of Müller glia from other animals were not dissimilar ( Table 5 ), ranging from passage 6-10 in species such as mouse, pig, human and whale, irrespective of the longevity or body size of each species ( Yegorov and Zelenin, 2003 ). However, variability in the passages before entering senescence was found in different cell-types, with human fibroblasts entering senescence at 20 passages ( Nogueira et al, 2021 ), human epithelial amniocytes entering senescence after only 3 passages ( Walen, 2004 ) and human Müller cells entering senescence at passage 6 ( Giannelli et al, 2011 ), similar to whale Müller glia. This relatively early entry into senescence could be a result of stress induced by the culture conditions used ( Liu et al, 2017 ).…”

“…Cytoskeletal changes during endothelial cell immortalization have been associated with the repression of cyclin-dependent kinase inhibitor 2A (p16INK4a), inducing random motility ( Kan et al, 2012 ). Other immortalized cell lines like human periodontal ligament fibroblast (hPLF-hTERT) also underwent changes related to immortalization that specifically affect cell migration, cell motility and cell adhesion ( Nogueira et al, 2021 ). Hence, the changes to the immortalized Müller cells witnessed here could be characteristic of the process of immortalization.…”