, Volume 19, Issue 1, pp 23–31 | Cite as

Mutual interactions between telomere heterogeneity and cell culture growth dynamics shape stochasticity of cell aging

  • Lucia Nanić
  • Nikolina Škrobot Vidaček
  • Sanda Ravlić
  • Eva Šatović
  • Miljenko Huzak
  • Ivica Rubelj
Research Article


Mathematical modeling and computational simulations are often used to explain the stochastic nature of cell aging. The models published thus far are based on the molecular mechanisms of telomere biology and how they dictate the dynamics of cell culture proliferation. However, the influence of cell growth conditions on telomere dynamics has been widely overlooked. These conditions include interactions with surrounding cells through contact inhibition, gradual accumulation of non-dividing cells, culture propagation and other cell culture maintenance factors. In order to follow the intrinsic growth dynamics of normal human fibroblasts we employed the fluorescent dye DiI and FACS analysis which can distinguish cells that undergo different numbers of divisions within culture. We observed rapid generation of cell subpopulations undergoing from 0 to 9 divisions within growing cultures at each passage analyzed. These large differences in number of divisions among individual cells guarantee a strong impact on generation of telomere length heterogeneity in normal cell cultures and suggest that culture conditions should be included in future modeling of cell senescence.


Telomere Senescence Cell culture Cell proliferation DiI SA-β-galactosidase 



We thank Mary Sopta for critically reading and editing the manuscript, Ela Kosor and Alenka Gagro for their participation in FACS analysis. We also thank Milena Ivanković, Marina Ferenac Kiš, Maja Matulić and Andrea Ćukušić Kalajžić for valuable discussions and practical assistance during the course of the experiments. This work was supported by Zaklada Adris.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10522_2017_9736_MOESM1_ESM.tif (1 mb)
Supplementary Fig. 7 [3H]thymidine labelling and SA-β Gal staining of NF fibroblasts at PD 10. a) Senescent cells are enlarged, they do not incorporate radioactivity and show strong SA-β Gal staining (purple arrow); young cells are smaller, 3H-T+ and do not show SA-β Gal staining (yellow arrow) b) Most cells were dividing and total of 97.25% incorporated radioactivity, among which 11.76% also showed traces of SA-β-Gal staining. > 1000 cells were counted for statistics (TIFF 1053 kb)
10522_2017_9736_MOESM2_ESM.tif (126 kb)
Supplementary Fig. 8 [3H]thymidine labelling index and SA-β Gal staining of NF fibroblasts at PDs 10 and 15. > 1000 cells were counted for statistics (TIFF 126 kb)


  1. Arino O, Kimmel M, Webb GF (1995) Mathematical modeling of the loss of telomere sequences. J Theor Biol 177:45–57CrossRefPubMedGoogle Scholar
  2. Arkus N (2005) A mathematical model of cellular apoptosis and senescence through the dynamics of telomere loss. J Theor Biol 235:13–32CrossRefPubMedGoogle Scholar
  3. Bourgeron T, Xu Z, Doumic M, Teresa Teixeira M (2015) The asymmetry of telomere replication contributes to replicative senescence heterogeneity. Sci Rep 5:15326CrossRefPubMedPubMedCentralGoogle Scholar
  4. Calado RT, Young NS (2009) Telomere diseases. N Engl J Med 361:2353–2365CrossRefPubMedPubMedCentralGoogle Scholar
  5. Calado R, Young N (2012) Telomeres in disease. F1000 Med Rep 4:8PubMedPubMedCentralGoogle Scholar
  6. Counter CM, Avilion AA, LeFeuvre CE, Stewart NG, Greider CW, Harley CB, Bacchetti S (1992) Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. EMBO J 11:1921–1929PubMedPubMedCentralGoogle Scholar
  7. Cukusic Kalajzic A, Škrobot Vidaček N, Huzak M, Ivankovic M, Rubelj I (2014) Telomere Q-PNA-FISH—reliable results from stochastic signals. PLoS ONE 9:e92559CrossRefPubMedPubMedCentralGoogle Scholar
  8. de Lange T (2002) Protection of mammalian telomeres. Oncogene 21:532–540CrossRefPubMedGoogle Scholar
  9. de Lange T, Zhu X-D, Küster B, Mann M, Petrini JHJ (2000) Cell-cycle-regulated association of RAD50/MRE11/NBS1 with TRF2 and human telomeres. Nat Genet 25:347–352CrossRefPubMedGoogle Scholar
  10. Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O et al (1995) A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA 92:9363–9367CrossRefPubMedPubMedCentralGoogle Scholar
  11. Ferenac M, Polancec D, Huzak M, Pereira-Smith O, Rubelj I (2005) Early-senescing human skin fibroblasts do not demonstrate accelerated telomere shortening. J Gerontol A Biol Sci Med Sci 60:820–829CrossRefPubMedGoogle Scholar
  12. Griffith JD, Comeau L, Rosenfield S, Stansel RM, Bianchi A, Moss H, de Lange T (1999) Mammalian telomeres end in a large duplex loop. Cell 97:503–514CrossRefPubMedGoogle Scholar
  13. Harley CB, Futcher AB, Greider CW (1990) Telomeres shorten during ageing of human fibroblasts. Nature 345:458–460CrossRefPubMedGoogle Scholar
  14. Hayflick L (1965) The limited in vitro lifetime of human diploid cell strains. Exp Cell Res 37:614–636CrossRefPubMedGoogle Scholar
  15. Hayflick L, Moorhead PS (1961) The serial cultivation of human diploid cell strains. Exp Cell Res 25:585–621CrossRefPubMedGoogle Scholar
  16. Hemann MT, Strong MA, Hao LY, Greider CW (2001) The shortest telomere, not average telomere length, is critical for cell viability and chromosome stability. Cell 107:67–77CrossRefPubMedGoogle Scholar
  17. Levy MZ, Allsopp RC, Futcher AB, Greider CW, Harley CB (1992) Telomere end-replication problem and cell aging. J Mol Biol 225:951–960CrossRefPubMedGoogle Scholar
  18. Nelson G, Wordsworth J, Wang C, Jurk D, Lawless C, Martin-Ruiz C, von Zglinicki T (2012) A senescent cell bystander effect: senescence-induced senescence. Aging Cell 11:345–349CrossRefPubMedPubMedCentralGoogle Scholar
  19. Olofsson P, Bertuch AA (2010) Modeling growth and telomere dynamics in Saccharomyces cerevisiae. J Theor Biol 263:353–359CrossRefPubMedGoogle Scholar
  20. Olofsson P, Kimmel M (1999) Stochastic models of telomere shortening. Math Biosci 158:75–92CrossRefPubMedGoogle Scholar
  21. Portugal RD, Land MGP, Svaiter BF (2008) A computational model for telomere-dependent cell-replicative aging. Biosystems 91:262–267CrossRefPubMedGoogle Scholar
  22. Proctor CJ, Kirkwood TBL (2002) Modelling telomere shortening and the role of oxidative stress. Mech Ageing Dev 123:351–363CrossRefPubMedGoogle Scholar
  23. Proctor CJ, Kirkwood TBL (2003) Modelling cellular senescence as a result of telomere state. Aging Cell 2:151–157CrossRefPubMedGoogle Scholar
  24. Rodier F, Campisi J (2011) Four faces of cellular senescence. J Cell Biol 192:547–556CrossRefPubMedPubMedCentralGoogle Scholar
  25. Rubelj I, Vondracek Z (1999) Stochastic mechanism of cellular aging—abrupt telomere shortening as a model for stochastic nature of cellular aging. J Theor Biol 197:425–438CrossRefPubMedGoogle Scholar
  26. Rubelj I, Huzak M, Brdar B (2000) Sudden senescence syndrome plays a major role in cell culture proliferation. Mech Ageing Dev 112:233–241CrossRefPubMedGoogle Scholar
  27. Rubelj I, Huzak M, Brdar B, Pereira-Smith OM (2002) A single-stage mechanism controls replicative senescence through Sudden Senescence Syndrome. Biogerontology 3:213–222CrossRefPubMedGoogle Scholar
  28. Shay JW, Wright WE (2001) Telomeres and telomerase: implications for cancer and aging. Radiat Res 155:188–193CrossRefPubMedGoogle Scholar
  29. Tan Z (1999) Intramitotic and intraclonal variation in proliferative potential of human diploid cells: explained by telomere shortening. J Theor Biol 198:259–268CrossRefPubMedGoogle Scholar
  30. Vidaček NŠ, Ćukušić A, Ivanković M, Fulgosi H, Huzak M, Smith JR, Rubelj I (2010) Abrupt telomere shortening in normal human fibroblasts. Exp Gerontol 45:235–242CrossRefPubMedGoogle Scholar
  31. von Zglinicki T (2002) Oxidative stress shortens telomeres. Trends Biochem Sci 27:339–344CrossRefGoogle Scholar
  32. Wu P, Takai H, de Lange T (2012) Telomeric 3′ overhangs derive from resection by Exo1 and Apollo and fill-in by POT1b-associated CST. Cell 150:39–52CrossRefPubMedPubMedCentralGoogle Scholar
  33. Xu Z, Duc KD, Holcman D, Teixeira MT (2013) The length of the shortest telomere as the major determinant of the onset of replicative senescence. Genetics 194:847–857CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2017

Authors and Affiliations

  1. 1.Department of Molecular BiologyRuder Boskovic InstituteZagrebCroatia
  2. 2.Department of MathematicsUniversity of ZagrebZagrebCroatia
  3. 3.Laboratory for Molecular and Cellular Biology, Division of Molecular BiologyRuder Boskovic InstituteZagrebCroatia

Personalised recommendations