• Open Access

Introducing a single-cell-derived human mesenchymal stem cell line expressing hTERT after lentiviral gene transfer

Authors

  • Wolfgang Böcker,

    1. Experimental Surgery and Regenerative Medicine, Department of Surgery, Ludwig-Maximilians-University (LMU), Nussbaumstrafie 20, 80336 Munich, Germany
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    • Shared first authorship

  • Zhanhai Yin,

    1. Experimental Surgery and Regenerative Medicine, Department of Surgery, Ludwig-Maximilians-University (LMU), Nussbaumstrafie 20, 80336 Munich, Germany
    2. Department of Orthopaedics, First Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, 710061 Xi'an, Shaanx Province, P. R. China
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    • Shared first authorship

  • Inga Drosse,

    1. Experimental Surgery and Regenerative Medicine, Department of Surgery, Ludwig-Maximilians-University (LMU), Nussbaumstrafie 20, 80336 Munich, Germany
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  • Florian Haasters,

    1. Experimental Surgery and Regenerative Medicine, Department of Surgery, Ludwig-Maximilians-University (LMU), Nussbaumstrafie 20, 80336 Munich, Germany
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  • Oliver Rossmann,

    1. Experimental Surgery and Regenerative Medicine, Department of Surgery, Ludwig-Maximilians-University (LMU), Nussbaumstrafie 20, 80336 Munich, Germany
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  • Matthias Wierer,

    1. Experimental Surgery and Regenerative Medicine, Department of Surgery, Ludwig-Maximilians-University (LMU), Nussbaumstrafie 20, 80336 Munich, Germany
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  • Cvetan Popov,

    1. Experimental Surgery and Regenerative Medicine, Department of Surgery, Ludwig-Maximilians-University (LMU), Nussbaumstrafie 20, 80336 Munich, Germany
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  • Melanie Locher,

    Corresponding author
    1. Center for Human Genetics and Laboratory Medicine, Lochhammerstr. 29, 82152 Munich, Germany
    • Correspondence to: Matthias SCHIEKER, MD,
      Experimental Surgery and Regenerative Medicine, Department of Surgery Ludwig-Maximilians-University Munich, Nussbaumstr. 20, D-80336 Munich, Germany.
      Tel.: +49 89 5160 7589
      Fax: +49 89 5160 5482
      Matthias.Schieker@med.uni-muenchen.de

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  • Wolf Mutschler,

    1. Experimental Surgery and Regenerative Medicine, Department of Surgery, Ludwig-Maximilians-University (LMU), Nussbaumstrafie 20, 80336 Munich, Germany
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  • Denitsa Docheva,

    1. Experimental Surgery and Regenerative Medicine, Department of Surgery, Ludwig-Maximilians-University (LMU), Nussbaumstrafie 20, 80336 Munich, Germany
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  • Matthias Schieker

    Corresponding author
    1. Experimental Surgery and Regenerative Medicine, Department of Surgery, Ludwig-Maximilians-University (LMU), Nussbaumstrafie 20, 80336 Munich, Germany
    • Correspondence to: Matthias SCHIEKER, MD,
      Experimental Surgery and Regenerative Medicine, Department of Surgery Ludwig-Maximilians-University Munich, Nussbaumstr. 20, D-80336 Munich, Germany.
      Tel.: +49 89 5160 7589
      Fax: +49 89 5160 5482
      Matthias.Schieker@med.uni-muenchen.de

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Abstract

Human mesenchymal stem cells (hMSCs) can be readily isolated from bone marrow and differentiate into multiple tissues, making them a promising target for future cell and gene therapy applications. The low frequency of hMSCs in bone marrow necessitates their isolation and expansion in vitro prior to clinical use, but due to senescence-associated growth arrest during culture, limited cell numbers can be generated. The lifespan of hMSCs has been extended by ectopic expression of human telomerase reverse transcriptase (hTERT) using retroviral vectors. Since malignant transformation was observed in hMSCs and retroviral vectors cause insertional mutagenesis, we ectopically expressed hTERT using lentiviral gene transfer. Single-cell-derived hMSC clones expressing hTERT did not show malignant transformation in vitro and in vivo after extended culture periods. There were no changes observed in the expression of tumour suppressor genes and karyotype. Cultured hMSCs lack telomerase activity, but it was significantly increased by ectopic expression of hTERT. HTERT expression prevented hMSC senescence and the cells showed significantly higher and unlimited proliferation capacity. Even after an extended culture period, hMSCs expressing hTERT preserved their stem cells character as shown by osteogenic, adipogenic and chon-drogenic differentiation. In summary, extending the lifespan of human mesenchymal stem cells by ectopic expression of hTERT using lentiviral gene transfer may be an attractive and safe way to generate appropriate cell numbers for cell and gene therapy applications.

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