Efficient immortalization of human dental pulp stem cells with expression of cell cycle regulators with the intact chromosomal condition

Abstract

Clinical studies have recently demonstrated that autologous transplantation of mobilized dental pulp stem cells is a safe and efficacious potential therapy for pulp regeneration. However, some limitations need to be addressed, such as the high cost of the safety and quality control tests for isolated individual dental pulp cell products before transplantation. Therefore, more efficient in vitro culturing of human dental pulp stem cells might be useful for providing low cost and high reliability testing for pulp regeneration therapy. In this study, we established a novel immortalized dental pulp stem cell line by co-expressing a mutant cyclin-dependent kinase 4 (CDK4 R24C), Cyclin D1, and telomerase reverse transcriptase (TERT). The established cell line maintained its original diploid chromosomes and stemness characteristics and exhibited an enhanced proliferation rate. In addition, we showed the immortalized human dental pulp stem cells still keeps their osteogenic and adipogenic differentiation abilities under appropriate culture conditions even though the cell proliferation was accelerated. Taken together, our established cell lines could serve as a useful in vitro tool for pulp regeneration therapy, and can contribute to reproducibility and ease of cell handling, thereby saving time and costs associated with safety and quality control tests.

Establishment and genetic characterization of cell lines derived from proliferating nasal polyps and sinonasal inverted papillomas

“…Results confirmed their individual origin (Table 1 ). Previously, cellular immortalization using mutant CDK4 and cyclin D1 in combination with TERT (K4DT) could generate immortalized cells with an intact chromosomal condition such as in human dental pulp stem cells, PT-5025 38 . However, this system sometimes generated immortalized cells with chromosomal abnormality.…”

Sinking Our Teeth in Getting Dental Stem Cells to Clinics for Bone Regeneration

“…This phenomenon hinders research efforts; thus, establishing immortalized cell lines of MSCs isolated from different body regions would make stem cell research more standardized and is therefore highly desirable [131]. Immortalized cell lines have been established from different kinds of human and mouse dental stem cells, with most researchers focusing on immortalizing DPSCs [132][133][134], including SHED [130,135], while less work has been done on dental epithelial stem cells (HERS/ERM) so far [136][137][138]. Most often, viral oncogenes (e.g., simian vacuolating virus 40 (SV40) large T-antigen or E6/E7) are used to immortalize the cells of interest [139].…”

“…Most often, viral oncogenes (e.g., simian vacuolating virus 40 (SV40) large T-antigen or E6/E7) are used to immortalize the cells of interest [139]. SV40 immortalization is effective in most cell types; it acts on the tumor suppressors p53 and Rb, while it also promotes the expression of the human telomerase reverse transcriptase (hTERT) to overcome telomer shortening [132,139]. However, viral oncogene immortalization is often associated with chromosome abnormalities, polyploidies, and changes in gene expression [132,139].…”

“…In other reports, it was shown that hTERT-immortalized SHED maintained differentiation capacity toward the neural linage, and no in vivo tumor formation was found, but abnormalities in soft agar colony formation and slight genetic instabilities were more pronounced [141]. In a more recent study, immortalized DPSCs were produced using an R24C mutant of cyclin-dependent kinase 4, cyclin D1, and TERT (K4DT-immortalization), since they reported that TERT alone was not sufficient for immortalization [132]. In this study, a stable chromosome pattern was shown, with an absence of senescence and a higher proliferation rate and differentiation capability in comparison to the primary cells in the investigated linage [132].…”

“…In a more recent study, immortalized DPSCs were produced using an R24C mutant of cyclin-dependent kinase 4, cyclin D1, and TERT (K4DT-immortalization), since they reported that TERT alone was not sufficient for immortalization [132]. In this study, a stable chromosome pattern was shown, with an absence of senescence and a higher proliferation rate and differentiation capability in comparison to the primary cells in the investigated linage [132]. Recently, the immortalization of SHED, pre-chosen cells with favorable stem cell characteristics such as octamer-binding transcription factors 3/4 (Oct3/4) expression and good reprogramming efficacy, was also reported using both HPV16-E7 and hTERT inserted by piggyBac transposon-based delivery [130].…”