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Lentiviral Transduction of Human Postnatal Skeletal (Stromal, Mesenchymal) Stem Cells: In Vivo Transplantation and Gene Silencing

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Abstract

Systems for gene transfer and silencing in human skeletal stem cells (hSSCs, also stromal or mesenchymal stem cells) are important for addressing critical issues in basic hSSC and skeletal biology and for developing gene therapy strategies for treatment of skeletal diseases. Whereas recent studies have shown the efficacy of lentiviral transduction for gene transfer in hSSCs in vitro, no study has yet proven that lentivector-transduced hSSCs retain their distinctive organogenic potential in vivo, as probed by in vivo transplantation assays. Therefore, in addition to analyzing the in vitro growth and differentiation properties of hSSCs transduced with advanced-generation lentivectors, we ectopically transplanted LV-eGFP-transduced hSSCs (along with an osteoconductive carrier) in the subcutaneous tissue of immunocompromised mice. eGFP-transduced cells formed heterotopic ossicles, generating osteoblasts, osteocytes, and stromal cells in vivo, which still expressed GFP at 2 months after transplantation. eGFP-expressing cells could be recovered from the ossicles 8 weeks posttransplantation and reestablished in culture as viable and proliferating cells. Further, we investigated the possibility of silencing individual genes in hSSCs using lentivectors encoding short hairpin precursors of RNA interfering sequences under the control of the Pol-III-dependent H1 promoter. Significant long-term silencing of both lamin A/C and GFP (an endogenous gene and a transgene, respectively) was obtained with lentivectors encoding shRNAs. These data provide the basis for analysis of the effect of gene knockdown during the organogenesis of bone in the in vivo transplantation system and for further studies on the silencing of alleles carrying dominant, disease-causing mutations.

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Acknowledgments

This study was supported by grants from MIUR, Telethon (grant GGP04263), AIRC, Istituto Superiore di Sanità and European Union (GENOSTEM, to P. B.) and from MIUR and Istituto Pasteur Cenci Bolognetti (to I. S.). We are grateful to V. Velotta and S. Morese for their contribution in the production of recombinant vectors.

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Authors and Affiliations

  1. Dipartimento di Genetica e Biologia Molecolare, Università di Roma La Sapienza, Rome, Italy

    S. Piersanti, G. Cherubini & I. Saggio

  2. Parco Scientifico Biomedico San Raffaele, Rome, Italy

    S. Piersanti, B. Sacchetti, A. Funari, S. Di Cesare, G. Cherubini, M. Riminucci, P. Bianco & I. Saggio

  3. Istituto Superiore di Sanità, Rome, Italy

    D. Bonci & C. Peschle

  4. Dipartimento di Medicina Sperimentale, Università dell’Aquila, L’Aquila, Italy

    M. Riminucci

  5. Dipartimento di Medicina Sperimentale e Patologia, Università La Sapienza, Rome, Italy

    P. Bianco

  6. Institute of Cell Biology and Pathology, CNR, Rome, Italy

    I. Saggio

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  1. S. Piersanti
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  2. B. Sacchetti
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  3. A. Funari
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Correspondence to P. Bianco.

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Piersanti, S., Sacchetti, B., Funari, A. et al. Lentiviral Transduction of Human Postnatal Skeletal (Stromal, Mesenchymal) Stem Cells: In Vivo Transplantation and Gene Silencing. Calcif Tissue Int 78, 372–384 (2006). https://doi.org/10.1007/s00223-006-0001-y

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