Journal of Bone and Mineral Metabolism

, Volume 29, Issue 2, pp 224–235 | Cite as

Human mesenchymal stem cell proliferation and osteogenic differentiation during long-term ex vivo cultivation is not age dependent

  • Stefan Fickert
  • Ute Schröter-Bobsin
  • Anna-Friederike Groß
  • Ute Hempel
  • Claudia Wojciechowski
  • Claudia Rentsch
  • Denis Corbeil
  • Klaus Peter Günther
Original Article

Abstract

Mesenchymal stem cells (MSCs) are of major clinical interest for the development of cell-based strategies to treat musculoskeletal diseases including critical-size bone defects caused by trauma, degenerative disorders, or infections. Elderly people mainly suffer from critical-size bone defects from the rising incidence of trauma, osteoporosis, and arthroplasties. In this study we investigated the influence of donor age on proliferation and osteogenic differentiation in long-term ex vivo cultures of primary human MSCs from patients in different age groups. Fifteen patients (8 men/7 women) comprised three age groups: (I) <50 years, (II) 50–65 years, and (III) >65 years. MSCs harvested from bone marrow derived from routine surgical procedures were isolated and cultured in standard medium over eight passages. Osteogenic differentiation was induced by dexamethasone (10 nM), ascorbic acid (300 μM), and β-glycerophosphate (3.5 mM). Osteogenic differentiation capacity of MSCs was quantified by alkaline phosphatase (ALP) activity, fluorescence-activated cell sorting (FACS) analysis of the surface markers CD9, CD90, CD54, CD166, CD105, CD44, and CD73, and RT-PCR for Coll I and II, Cbfa 1, ALP, OC, BSP1, and GAPDH genes characterized the phenotypic changes during monolayer expansion. In vitro chondrogenic differentiation was analyzed by immunohistochemistry and RT-PCR. Progenitor cells could be expanded in the long term from all bone marrow donations. FACS single staining analysis from MSCs showed no significant difference between the age groups. The surface antigen CD166 was predominantly found in all cell cultures independently of differentiation stage. Comparison of expanded and differentiated MSCs within a single age group showed that undifferentiated MSCs had higher CD44 levels. Osteogenic stimulation of MSCs was confirmed by measuring ALP activity. The highest ALP activity was found in probands of the age group >65 years. Additionally, we observed a tendency toward male-specific ALP increase during differentiation. Osteogenic marker gene expression in MSCs was detected by RT-PCR. No significant expression differences were detected between the three donor age groups. Micromass culture of MSCs resulted histologically and immunohistologically in a chondrogenic phenotype. Elderly osteoprogenitor cell donors are a highly clinically relevant patient population. In summary, cultivation leads to a reduced osteogenic differentiation capacity regardless of age. Because donor age does not affect osteogenic differentiation potential, it should not be used as an exclusion criterion for autologous transplantation of human adult MSCs.

Keywords

Mesenchymal stem cell Osteogenic differentiation Age 

References

  1. 1.
    Bianco P, Robey PG (2001) Stem cells in tissue engineering. Nature (Lond) 414:118–121CrossRefGoogle Scholar
  2. 2.
    Ringe J, Kaps C, Burmester GR, Sittinger M (2002) Stem cells for regenerative medicine: advances in the engineering of tissues and organs. Naturwissenschaften 89:338–351PubMedCrossRefGoogle Scholar
  3. 3.
    Polak J, Hench L (2005) Gene therapy progress and prospects: in tissue engineering. Gene Ther 12:1725–1733PubMedCrossRefGoogle Scholar
  4. 4.
    Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop D, Horwitz E (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8:315–317PubMedCrossRefGoogle Scholar
  5. 5.
    Nishida S, Endo N, Yamagiwa H, Tanizawa T, Takahashi HE (1999) Number of osteoprogenitor cells in human bone marrow markedly decreases after skeletal maturation. J Bone Miner Metab 17:171–177PubMedCrossRefGoogle Scholar
  6. 6.
    Muschler GF, Nitto H, Boehm CA, Easley KA (2001) Age- and gender-related changes in the cellularity of human bone marrow and the prevalence of osteoblastic progenitors. J Orthop Res 19:117–125PubMedCrossRefGoogle Scholar
  7. 7.
    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 (NY) 33:919–926Google Scholar
  8. 8.
    DIppolito G, Schiller PC, Ricordi C, Roos BA, Howard GA (1999) Age-related osteogenic potential of mesenchymal stromal stem cells from human vertebral bone marrow. J Bone Miner Res 14:1115–1122CrossRefGoogle Scholar
  9. 9.
    Martinez ME, del Campo MT, Medina S, Sanchez M, Sanchez-Cabezudo MJ, Esbrit P, Martinez P, Moreno I, Rodrigo A, Garces MV, Munuera L (1999) Influence of skeletal site of origin and donor age on osteoblastic cell growth and differentiation. Calcif Tissue Int 64:280–286PubMedCrossRefGoogle Scholar
  10. 10.
    Muraglia A, Cancedda R, Quarto R (2002) Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model. J Cell Sci 113:1161–1166Google Scholar
  11. 11.
    Justesen J, Stenderup K, Eriksen EF, Kassem M (2002) Maintenance of osteoblastic and adipocytic differentiation potential with age and osteoporosis in human marrow stromal cell cultures. Calcif Tissue Int 71:36–44PubMedCrossRefGoogle Scholar
  12. 12.
    Leskela HV, Risteli J, Niskanen S, Koivunen J, Ivaska KK, Lehenkari P (2003) Osteoblast recruitment from stem cells does not decrease by age at late adulthood. Biochem Biophys Res Commun 311:1008–1013PubMedCrossRefGoogle Scholar
  13. 13.
    Schecroun N, Delloye C (2003) Bone-like nodules formed by human bone marrow stromal cells: comparative study and characterization. Bone (NY) 32:252–260Google Scholar
  14. 14.
    Abdallah BM, Haack-Sorensen M, Fink T, Kassem M (2006) Inhibition of osteoblast differentiation but not adipocyte differentiation of mesenchymal stem cells by sera obtained from aged females. Bone (NY) 39:181–188Google Scholar
  15. 15.
    Phinney DG, Kopen G, Righter W, Webster S, Tremain N, Prockop DJ (1999) Donor variation in the growth properties and osteogenic potential of human marrow stromal cells. J Cell Biochem 75:424–436PubMedCrossRefGoogle Scholar
  16. 16.
    Ciapetti G, Ambrosio L, Marletta G, Baldini N, Giunti A (2006) Human bone marrow stromal cells: in vitro expansion and differentiation for bone engineering. Biomaterials 27:6150–6160PubMedCrossRefGoogle Scholar
  17. 17.
    Bonab MM, Alimoghaddam K, Talebian F, Ghaffari SH, Ghavamzadeh A, Nikbin B (2006) Aging of mesenchymal stem cell in vitro. BMC Cell Biol 7:14PubMedCrossRefGoogle Scholar
  18. 18.
    Moussavi-Harami F, Duwayri Y, Martin JA, Buckwalter JA (2004) Oxygen effects on senescence in chondrocytes and mesenchymal stem cells: consequences for tissue engineering. Iowa Orthop J 24:15–20PubMedGoogle Scholar
  19. 19.
    Pittenger MF, Martin BJ (2004) Mesenchymal stem cells and their potential as cardiac therapeutics. Circ Res 95:9–20PubMedCrossRefGoogle Scholar
  20. 20.
    Mareschi K, Ferrero I, Rustichelli D, Aschero S, Gammaitoni L, Aglietta M, Madon E, Fagioli F (2006) Expansion of mesenchymal stem cells isolated from pediatric and adult donor bone marrow. J Cell Biochem 97:744–754PubMedCrossRefGoogle Scholar
  21. 21.
    Foster LJ, Zeemann PA, Li C, Mann M, Jensen ON, Kassem M (2005) Differential expression profiling of membrane proteins by quantitative proteomics in a human mesenchymal stem cell line undergoing osteoblast differentiation. Stem Cells 23:1367–1377PubMedCrossRefGoogle Scholar
  22. 22.
    Aubin JE, Liu F, Malaval L, Gupta AK (1995) Osteoblast and chondroblast differentiation. Bone (NY) 17:77S–83SGoogle Scholar
  23. 23.
    Zhu H, Mitsuhashi N, Klein A, Barsky LW, Weinberg K, Barr ML, Demetriou A, Wu GD (2006) The role of the hyaluronan receptor CD44 in mesenchymal stem cell migration in the extracellular matrix. Stem Cells 24:928–935PubMedCrossRefGoogle Scholar
  24. 24.
    Aoyama K, Oritani K, Yokota T, Ishikawa J, Nishiura T, Miyake K, Kanakura Y, Tomiyama Y, Kincade PW, Matsuzawa Y (1999) Stromal cell CD9 regulates differentiation of hematopoietic stem/progenitor cells. Blood 93:2586–2594PubMedGoogle Scholar
  25. 25.
    Marom R, Shur I, Solomon R, Benayahu D (2005) Characterization of adhesion and differentiation markers of osteogenic marrow stromal cells. J Cell Physiol 202:41–48PubMedCrossRefGoogle Scholar
  26. 26.
    Katzburg S, Lieberherr M, Ornoy A, Klein BY, Hendel D, Somjen D (1999) Isolation and hormonal responsiveness of primary cultures of human bone-derived cells: gender and age differences. Bone (NY) 25:667–673Google Scholar
  27. 27.
    Liu F, Malaval L, Aubin JE (2003) Global amplification polymerase chain reaction reveals novel transitional stages during osteoprogenitor differentiation. J Cell Sci 116:1787–1796PubMedCrossRefGoogle Scholar

Copyright information

© The Japanese Society for Bone and Mineral Research and Springer 2010

Authors and Affiliations

  • Stefan Fickert
    • 1
  • Ute Schröter-Bobsin
    • 2
  • Anna-Friederike Groß
    • 2
  • Ute Hempel
    • 3
  • Claudia Wojciechowski
    • 2
  • Claudia Rentsch
    • 4
  • Denis Corbeil
    • 5
  • Klaus Peter Günther
    • 2
  1. 1.Orthopaedic and Trauma Surgery CenterUniversity Medical Center MannheimMannheimGermany
  2. 2.Department of Orthopaedic Surgery, Medical Faculty of the Technical University DresdenUniversity Hospital Carl Gustav Carus DresdenDresdenGermany
  3. 3.Institute of Physiological ChemistryMedical Faculty of the Technical University DresdenDresdenGermany
  4. 4.Department of Trauma and Reconstructive Surgery, University Hospital Carl Gustav Carus DresdenMedical Faculty of the Technical University DresdenDresdenGermany
  5. 5.Tissue Engineering Laboratories, BIOTECTechnical University DresdenDresdenGermany

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