Molecular Biology Reports

, Volume 38, Issue 8, pp 5161–5168 | Cite as

Replicative senescence of human bone marrow and umbilical cord derived mesenchymal stem cells and their differentiation to adipocytes and osteoblasts

  • HuanChen Cheng
  • Lin Qiu
  • Jun MaEmail author
  • Hao Zhang
  • Mei Cheng
  • Wei Li
  • Xuefei Zhao
  • Keyu Liu


Mesenchymal stem cells (MSC) which have self-renewal and multiple differentiation potential in vitro play important roles in regenerative medicine and tissue engineering. However, long-term culture in vitro leads to senescence which results in the growth arrest and reduction of differentiation. In this study, MSC derived from human bone-marrow (BM-MSC) and umbilical cord (UC-MSC) were cultured in vitro lasted to senescence. Senescence and apoptosis detection showed that the senescent cells increased significantly but the increase of apoptosis was not significant in the long term culture. Senescence related genes p16, p21 and p53 increased gradually in BM-MSC. However, p16 and p53 reduced and then increased but with the gradual increase of p21 in UC-MSC. Adipogenic differentiation decreased whereas the propensity for osteogenic differentiation increased in senescent MSC. Real time RT-PCR demonstrated that both C/EBPα and PPARγ decreased in senescent BM-MSC. However, in UC-MSC, PPARγ decreased but C/EBPα increased in late phase compared to early phase. The study demonstrated p21 was important in the senescence of BM-MSC and UC-MSC. C/EBPα and PPARγ could regulate the balance of adipogenic differentiation in BM-MSC but only PPARγ not C/EBPα was involved in the adipogenic differentiation in UC-MSC.


MSC Adipogenic Osteogenic Senescence Gene 



The study was supported by Harbin Technology and Innovation Foundation for Youth (Grant numbers 2010RFQQS082 and 2009RFQQS027).


  1. 1.
    Chen Y, Shao JZ, Xiang LX, Dong XJ, Zhang GR (2008) Mesenchymal stem cells: a promising candidate in regenerative medicine. Int J Biochem Cell Biol 40:815–820PubMedCrossRefGoogle Scholar
  2. 2.
    Mimeault M, Batra SK (2006) Concise review: recent advances on the significance of stem cells in tissue regeneration and cancer therapies. Stem Cells 24:2319–2345PubMedCrossRefGoogle Scholar
  3. 3.
    Zhou S, Greenberger JS, Epperly MW, Goff JP, Adler C, Leboff MS, Glowacki J (2008) Age-related intrinsic changes in human bone-marrow-derived mesenchymal stem cells and their differentiation to osteoblasts. Aging Cell 7:335–343PubMedCrossRefGoogle Scholar
  4. 4.
    Ksiazek K (2009) A comprehensive review on mesenchymal stem cell growth and senescence. Rejuvenation Res 12:105–116PubMedCrossRefGoogle Scholar
  5. 5.
    Kiyono T, Foster SA, Koop JI, McDougall JK, Galloway DA, Klingelhutz AJ (1998) Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells. Nature 396:84–88PubMedCrossRefGoogle Scholar
  6. 6.
    Stewart SA, Ben-Porath I, Carey VJ, O’Connor BF, Hahn WC, Weinberg RA (2003) Erosion of the telomeric single-strand overhang at replicative senescence. Nat Genet 33:492–496PubMedCrossRefGoogle Scholar
  7. 7.
    von Zglinicki T (2002) Oxidative stress shortens telomeres. Trends Biochem Sci 27:339–344CrossRefGoogle Scholar
  8. 8.
    Park JS, Kim HY, Kim HW, Chae GN, Oh HT, Park JY, Shim H, Seo M, Shin EY, Kim EG, Park SC, Kwak SJ (2005) Increased caveolin-1, a cause for the declined adipogenic potential of senescent human mesenchymal stem cells. Mech Ageing Dev 126:551–559PubMedCrossRefGoogle Scholar
  9. 9.
    Terai M, Uyama T, Sugiki T, Li XK, Umezawa A, Kiyono T (2005) Immortalization of human fetal cells: the life span of umbilical cord blood-derived cells can be prolonged without manipulating p16INK4a/RB braking pathway. Mol Biol Cell 16:1491–1499PubMedCrossRefGoogle Scholar
  10. 10.
    Heo JY, Jing K, Song KS, Seo KS, Park JH, Kim JS, Jung YJ, Hur GM, Jo DY, Kweon GR, Yoon WH, Lim K, Hwang BD, Jeon BH, Park JI (2009) Downregulation of APE1/Ref-1 is involved in the senescence of mesenchymal stem cells. Stem Cells 27:1455–1462Google Scholar
  11. 11.
    Stenderup K, Justesen J, Clausen C, Kassem M (2003) Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells. Bone 33:919–926PubMedCrossRefGoogle Scholar
  12. 12.
    Hayflickl (1965) The limited in vitro lifetime of human diploid cell strains. Exp Cell Res 37:614–636CrossRefGoogle Scholar
  13. 13.
    Galderisi U, Helmbold H, Squillaro T, Alessio N, Komm N, Khadang B, Cipollaro M, Bohn W, Giordano A (2009) In vitro senescence of rat mesenchymal stem cells is accompanied by downregulation of stemness-related and DNA damage repair genes. Stem Cells Dev 18:1033–1042PubMedCrossRefGoogle Scholar
  14. 14.
    Raz V, Vermolen BJ, Garini Y, Onderwater JJ, Mommaas-Kienhuis MA, Koster AJ, Young IT, Tanke H, Dirks RW (2008) The nuclear lamina promotes telomere aggregation and centromere peripheral localization during senescence of human mesenchymal stem cells. J Cell Sci 121:4018–4028PubMedCrossRefGoogle Scholar
  15. 15.
    Herbig U, Jobling WA, Chen BP, Chen DJ, Sedivy JM (2004) Telomere shortening triggers senescence of human cells through a pathway involving ATM, p53, and p21(CIP1), but not p16(INK4a). Mol Cell 14:501–513PubMedCrossRefGoogle Scholar
  16. 16.
    Jin Y, Kato T, Furu M, Nasu A, Kajita Y, Mitsui H, Ueda M, Aoyama T, Nakayama T, Nakamura T, Toguchida J (2010) Mesenchymal stem cells cultured under hypoxia escape from senescence via down-regulation of p16 and extracellular signal regulated kinase. Biochem Biophys Res Commun 391:1471–1476PubMedCrossRefGoogle Scholar
  17. 17.
    Shibata KR, Aoyama T, Shima Y, Fukiage K, Otsuka S, Furu M, Kohno Y, Ito K, Fujibayashi S, Neo M, Nakayama T, Nakamura T, Toguchida J (2007) Expression of the p16INK4A gene is associated closely with senescence of human mesenchymal stem cells and is potentially silenced by DNA methylation during in vitro expansion. Stem Cells 25:2371–2382PubMedCrossRefGoogle Scholar
  18. 18.
    Banfi A, Muraglia A, Dozin B, Mastrogiacomo M, Cancedda R, Quarto R (2000) Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: implications for their use in cell therapy. Exp Hematol 28:707–715PubMedCrossRefGoogle Scholar
  19. 19.
    Kretlow JD, Jin YQ, Liu W, Zhang WJ, Hong TH, Zhou G, Baggett LS, Mikos AG, Cao Y (2008) Donor age and cell passage affects differentiation potential of murine bone marrow-derived stem cells. BMC Cell Biol 28:60CrossRefGoogle Scholar
  20. 20.
    Wagner W, Horn P, Castoldi M, Diehlmann A, Bork S, Saffrich R, Benes V, Blake J, Pfister S, Eckstein V, Ho AD (2008) Replicative senescence of mesenchymal stem cells: a continuous and organized process. PLoS One 3:e2213PubMedCrossRefGoogle Scholar
  21. 21.
    Fux C, Mitta B, Kramer BP, Fussenegger M (2004) Dual-regulated expression of C/EBP-alpha and BMP-2 enables differential differentiation of C2C12 cells into adipocytes and osteoblasts. Nucleic Acids Res 32:e1PubMedCrossRefGoogle Scholar
  22. 22.
    Schoonjans K, Staels B, Auwerx J (1996) The peroxisome proliferator activated receptors (PPARS) and their effects on lipid metabolism and adipocyte differentiation. Biochim Biophys Acta 1302:93–109PubMedGoogle Scholar
  23. 23.
    Fan Q, Tang T, Zhang X, Dai K (2009) The role of CCAAT/enhancer binding protein (C/EBP)-alpha in osteogenesis of C3H10T1/2 cells induced by BMP-2. J Cell Mol Med 13:2489–2505PubMedCrossRefGoogle Scholar
  24. 24.
    Giordano A, Galderisi U, Marino IR (2007) From the laboratory bench to the patient’s bedside: an update on clinical trials with mesenchymal stem cells. J Cell Physiol 211:27–35PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • HuanChen Cheng
    • 1
  • Lin Qiu
    • 1
  • Jun Ma
    • 1
    Email author
  • Hao Zhang
    • 1
  • Mei Cheng
    • 1
  • Wei Li
    • 1
  • Xuefei Zhao
    • 1
  • Keyu Liu
    • 1
  1. 1.Institute of Harbin Hematology & OncologyHarbin First HospitalHarbinPeople’s Republic of China

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