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Proliferation and differentiation of human adipose-derived mesenchymal stem cells (ASCs) into osteoblastic lineage are passage dependent

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Abstract

Objective

The effect of in vitro expansion of human adipose-derived stem cells (ASCs) on stem cell properties is controversial. We examined serial subcultivation with expansion on the ability of ASCs to grow and differentiate into osteoblastic lineages.

Design

Flow cytometric analysis, growth kinetics, cell population doubling time, light microscopy and confocal analysis, and osteogenesis induction were performed to assess growth and osteogenic potential of subcultivated ASCs at passages 2 (P2), P4 and P6.

Results

Flow cytometric analysis revealed that ASCs at P2 express classical mesenchymal stem cell markers including CD44, CD73, and CD105, but not CD14, CD19, CD34, CD45, or HLA-DR. Calcium deposition and alkaline phosphatase activity were the highest at P2 but completely abrogated at P4. Increased passage number impaired cell growth; P2 cultures exhibited exponential growth, while cells at P4 and P6 showed near linear growth with cell population doubling times increased from 3.2 at P2 to 4.8 d at P6. Morphologically, cells in various subcultivation stages showed flattened shape at low density but spindle-like structures at confluency as judged by phalloidin staining.

Conclusions

Osteogenic potential of ASCs is impaired by successive passaging and may not serve as a useful clinical source of osteogenic ASCs past P2.

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Acknowledgments

WHF and JDB designed and coordinated the study. JDB, FS and EA provided the human fat tissues. WS, OK, EH, DA, SM, BB and RAM carried out the experiments. WF and JDB wrote and critically revised the manuscript. EH helped with the conception and design of the study, and with drafting the manuscript. All authors approved the final manuscript.

Conflict of interest

All authors declare that they have no competing interests. Jiovanni A. Di Battista is a professor of Medicine––Department of Pharmacology and Therapeutics at McGill University and recipient of a Canadian Institute of Health Research Grant (CIHR). Dr. Wissam Faour is a recipient of a Lebanese National Council for Scientific Research (LNCSR) Grant and an Assistant Professor of Pharmacology at the School of Medicine at the Lebanese American University. This work is supported by a grant from the Lebanese National Council for Scientific Research (LNCSR) and the Canadian Institute of Health research (CIHR).

Author information

Authors and Affiliations

  1. Division of Rheumatology, Royal Victoria Hospital, McGill University Health Center, Montreal, QC, Canada

    Jiovanni A. Di Battista & Ozge Kizilay

  2. Division of Experimental Medicine, Department of Medicine, McGill University, Montreal, QC, Canada

    Jiovanni A. Di Battista & Ozge Kizilay

  3. Department of Natural Sciences, School of Arts and Sciences, Lebanese American University, Byblos, Lebanon

    Wassim Shebaby

  4. Department of Chemistry and Biochemistry, Faculty of Sciences I, Lebanese University, Hadath, Lebanon

    Eva Hamade

  5. Génomique et Santé, Lebanese University, Hadath, Lebanon

    Raghida Abou Merhi

  6. Université de Lyon, Lyon, 69361, France

    Saida Mebarek

  7. Université Lyon 1, Villeurbanne, 69622, France

    Saida Mebarek

  8. INSA-Lyon, Villeurbanne, 69622, France

    Saida Mebarek

  9. CPE Lyon, Villeurbanne, 69616, France

    Saida Mebarek

  10. ICBMS CNRS UMR 5246, Villeurbanne, 69622, France

    Saida Mebarek

  11. Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, ICBMS UMR 5246-Université Lyon 1. Platforme de recherche et d’analyse en Sciences de l’environnement, Université Libanaise, Beirut, Lebanon

    Dina Abdallah

  12. Department of Biochemistry, Laboratory of Immunology, EDST-PRASE, Lebanese University, Faculty of Sciences, Hadath-Beirut, Lebanon

    Bassam Badran

  13. Hopital Saint-Louis, Jounieh, Lebanon

    Fady Saad

  14. Department of Surgery, Lebanese American University Medical Center-Rizk Hospital, Beirut, Lebanon

    Eddie K. Abdalla

  15. Gilbert and Rose-Marie Chagoury School of Medicine, Lebanese American University, 36, Byblos, Lebanon

    Wissam H. Faour

Authors
  1. Jiovanni A. Di Battista
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  2. Wassim Shebaby
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  3. Ozge Kizilay
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  4. Eva Hamade
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  5. Raghida Abou Merhi
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  6. Saida Mebarek
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  7. Dina Abdallah
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  8. Bassam Badran
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  9. Fady Saad
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  10. Eddie K. Abdalla
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  11. Wissam H. Faour
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Corresponding author

Correspondence to Wissam H. Faour.

Additional information

Responsible Editor: John Di Battista.

W. Shebaby, O. Kizilay and E. Hamade contributed equally to this work.

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Di Battista, J.A., Shebaby, W., Kizilay, O. et al. Proliferation and differentiation of human adipose-derived mesenchymal stem cells (ASCs) into osteoblastic lineage are passage dependent. Inflamm. Res. 63, 907–917 (2014). https://doi.org/10.1007/s00011-014-0764-y

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  • DOI: https://doi.org/10.1007/s00011-014-0764-y

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