Telomeres are repetitive nucleoprotein DNA sequences that shorten with each cell division. The stem cells activate telomerase to compensate for the telomere loss. This study aimed to evaluate the effect of cultivation passaging on the relative telomere length and proliferation capacity of dental pulp stem cells. We used ten dental pulp stem cell (DPSC) lineages stored for 12 months using uncontrolled-rate freezing to reach the study's goal. We analyzed their proliferation rate, phenotype using flow cytometry, multipotency, and relative telomere length using a qPCR analysis. We determined the relative telomere length in the added study by performing analysis after one, two, and three weeks of cultivation with no passaging. We documented the telomere attrition with increasing passaging. The shorter the relative telomere length, the lower reached population doublings, and longer population doubling time were observed at the end of the cultivation. We observed the telomere prolongation in DPSCs cultivated for two weeks with no passaging in the added subsequent study. We concluded that excessive proliferation demands on DPSCs during in vitro cultivation result in telomere attrition. We opened the theory that the telomerase might be more efficient during cell cultivation with no passaging. This observation could help in preserving the telomere length during ex vivo DPSC expansion.

Department of Dentistry Charles University Faculty of Medicine in Hradec Kralove and University Hospital Hradec Kralove 500 05 Hradec Kralove Czech Republic

Department of Histology and Embryology Charles University Faculty of Medicine in Hradec Kralove 500 03 Hradec Kralove Czech Republic

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Krebsbach P.H., Villa-Diaz L.G. The Role of Integrin alpha6 (CD49f) in Stem Cells: More than a Conserved Biomarker. Stem Cells Dev. 2017;26:1090–1099. doi: 10.1089/scd.2016.0319. PubMed DOI PMC

Bischoff D.S., Makhijani N.S., Yamaguchi D.T. Constitutive expression of human telomerase enhances the proliferation potential of human mesenchymal stem cells. Biores. Open Access. 2012;1:273–279. doi: 10.1089/biores.2012.0252. PubMed DOI PMC

Hayflick L. The limited in vitro lifetime of human diploid cell strains. Exp. Cell Res. 1965;37:614–636. doi: 10.1016/0014-4827(65)90211-9. PubMed DOI

Serakinci N., Graakjaer J., Kolvraa S. Telomere stability and telomerase in mesenchymal stem cells. Biochimie. 2008;90:33–40. doi: 10.1016/j.biochi.2007.09.005. PubMed DOI

de Lange T. Shelterin: The protein complex that shapes and safeguards human telomeres. Genes Dev. 2005;19:2100–2110. doi: 10.1101/gad.1346005. PubMed DOI

de Lange T. T-loops and the origin of telomeres. Nat. Rev. Mol. Cell Biol. 2004;5:323–329. doi: 10.1038/nrm1359. PubMed DOI

Flores I., Benetti R., Blasco M.A. Telomerase regulation and stem cell behaviour. Curr. Opin. Cell Biol. 2006;18:254–260. doi: 10.1016/j.ceb.2006.03.003. PubMed DOI

Sharpless N.E., DePinho R.A. How stem cells age and why this makes us grow old. Nat. Rev. Mol. Cell Biol. 2007;8:703–713. doi: 10.1038/nrm2241. PubMed DOI

Aubert G. Telomere dynamics and aging. Prog. Mol. Biol. Transl. Sci. 2014;125:89–111. doi: 10.1016/B978-0-12-397898-1.00004-9. PubMed DOI

Drela K., Stanaszek L., Nowakowski A., Kuczynska Z., Lukomska B. Experimental Strategies of Mesenchymal Stem Cell Propagation: Adverse Events and Potential Risk of Functional Changes. Stem Cells Int. 2019;2019:7012692. doi: 10.1155/2019/7012692. PubMed DOI PMC

Mokry J., Soukup T., Micuda S., Karbanova J., Visek B., Brcakova E., Suchanek J., Bouchal J., Vokurkova D., Ivancakova R. Telomere attrition occurs during ex vivo expansion of human dental pulp stem cells. J. Biomed. Biotechnol. 2010;2010:673513. doi: 10.1155/2010/673513. PubMed DOI PMC

Pilbauerova N., Soukup T., Suchankova Kleplova T., Suchanek J. Enzymatic Isolation, Amplification and Characterization of Dental Pulp Stem Cells. Folia Biol. 2019;65:124–133. PubMed

Suchanek J., Visek B., Soukup T., El-Din Mohamed S.K., Ivancakova R., Mokry J., Aboul-Ezz E.H., Omran A. Stem cells from human exfoliated deciduous teeth--isolation, long term cultivation and phenotypical analysis. Acta Med. 2010;53:93–99. doi: 10.14712/18059694.2016.66. PubMed DOI

Cawthon R.M. Telomere measurement by quantitative PCR. Nucleic Acids Res. 2002;30:e47. doi: 10.1093/nar/30.10.e47. PubMed DOI PMC

Jeon B.G., Kang E.J., Kumar B.M., Maeng G.H., Ock S.A., Kwack D.O., Park B.W., Rho G.J. Comparative analysis of telomere length, telomerase and reverse transcriptase activity in human dental stem cells. Cell Transplant. 2011;20:1693–1705. doi: 10.3727/096368911X565001. PubMed DOI

Lai T.P., Wright W.E., Shay J.W. Comparison of telomere length measurement methods. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2018;373 doi: 10.1098/rstb.2016.0451. PubMed DOI PMC

Suchanek J., Kleplova T.S., Kapitan M., Soukup T. The effect of fetal calf serum on human dental pulp stem cells. Acta Med. 2013;56:142–149. doi: 10.14712/18059694.2014.9. PubMed DOI

Alraies A., Alaidaroos N.Y., Waddington R.J., Moseley R., Sloan A.J. Variation in human dental pulp stem cell ageing profiles reflect contrasting proliferative and regenerative capabilities. BMC Cell Biol. 2017;18:12. doi: 10.1186/s12860-017-0128-x. PubMed DOI PMC

Gronthos S., Brahim J., Li W., Fisher L.W., Cherman N., Boyde A., DenBesten P., Robey P.G., Shi S. Stem cell properties of human dental pulp stem cells. J. Dent. Res. 2002;81:531–535. doi: 10.1177/154405910208100806. PubMed DOI

Al-Saqi S.H., Saliem M., Quezada H.C., Ekblad Å., Jonasson A.F., Hovatta O., Götherström C. Defined serum- and xeno-free cryopreservation of mesenchymal stem cells. Cell Tissue Bank. 2015;16:181–193. doi: 10.1007/s10561-014-9463-8. PubMed DOI

Bailey S.M., Brenneman M.A., Goodwin E.H. Frequent recombination in telomeric DNA may extend the proliferative life of telomerase-negative cells. Nucleic Acids Res. 2004;32:3743–3751. doi: 10.1093/nar/gkh691. PubMed DOI PMC

Hu H., Li B., Duan S. The Alteration of Subtelomeric DNA Methylation in Aging-Related Diseases. Front Genet. 2018;9:697. doi: 10.3389/fgene.2018.00697. PubMed DOI PMC

Banfi A., Muraglia A., Dozin B., Mastrogiacomo M., Cancedda R., Quarto R. Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: Implications for their use in cell therapy. Exp. Hematol. 2000;28:707–715. doi: 10.1016/S0301-472X(00)00160-0. PubMed DOI

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