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Z Orthop Ihre Grenzgeb 2005; 143(6): 677-683
DOI: 10.1055/s-2005-872526
Grundlagenforschung

© Georg Thieme Verlag Stuttgart · New York

Kontrollierter Gentransfer in humane adulte mesenchymale Stammzellen mittels adenoviraler Vektoren

Refined Adenoviral Transduction for Controlled Gene Transfer into Human Adult Mesenchymal Stem CellsH. Koch1 , J. A. Jadlowiec2 , 3 , J. D. Whalen4 , C. Lattermann5 , P. Robbins6 , F. H. Fu5 , H. Merk1 , J. O. Hollinger2
  • 1Klinik für Orthopädie und orthopädische Chirurgie, Universität Greifswald
  • 2Bone Tissue Engineering Center, Carnegie Mellon University (CMU), Pittsburgh, USA
  • 3Department of Biological Sciences, CMU, Pittsburgh, USA
  • 4Shadyside Hospital, University of Pittsburgh Medical Center (UPMC), Pittsburgh, USA
  • 5Department of Orthopaedic Surgery, UPMC, Pittsburgh, USA
  • 6Department of Molecular Genetics and Biochemistry, University of Pittsburgh, USA
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Publication History

Publication Date:
28 December 2005 (online)

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Zusammenfassung

Studienziel: Der adenoviral vermittelte Gentransfer bleibt ein wichtiges Instrument für die Grundlagenforschung. Unsere Hypothese lautete: Die adenovirale Transduktion von humanen mesenchymalen Stammzellen (hMSC) in vitro kann durch eine kontrollierte Anwendung der Parameter Viruslast (MOI) und Expositionsdauer verbessert werden. Methode: Um unsere Hypothese zu testen, wurden hMSC mit adenoviralen Vektoren für Luciferase und BMP-2 transduziert. Verschiedene MOI und Expositionszeiten wurden angewandt, die Produktion des Transgens und Gesamtproteingehalt wurden bestimmt. Um die praktische Relevanz zu beurteilen, quantifizierten wir die mRNA der Knochenmarkergene Typ-1-Kollagen und Runx2 mittels quantitativer real-time PCR. Als phänotypischen Knochenmarker bestimmten wir die alkalische Phosphataseaktivität. Die statistische Auswertung erfolgte mittels Varianzanalyse und Post-hoc-Tests (p < 0,05). Ergebnisse: Längere Exposition führte zu abnehmenden Mengen des Transgens und des Gesamtproteingehaltes. Eine zunehmende MOI hatte bei Exposition bis zu 4 Stunden einen Anstieg der Transgenproduktion zur Folge. Der Transfer des BMP-2-Gens führte zu osteoblastärer Differenzierung als Hinweis auf die biologische Aktivität des Transgens. Schlussfolgerung: Die Expositionsdauer ist für die Toxizität von entscheidender Bedeutung und sollte für hMSC 4 Stunden nicht überschreiten. Obwohl zunehmende Expositionsdauer zum Zelluntergang führt, scheinen dies die überlebenden Zellen über eine gesteigerte Produktion des Transgens teilweise kompensieren zu können. Hieraus ergibt sich, dass die Transduktionseffizienz nicht eindeutig nach einem Ja-oder-Nein-Schema beurteilt werden kann.

Abstract

Aim: Adenoviral gene transfer remains a powerful tool for basic research purposes. We hypothesize that adenoviral transduction of human mesenchymal stem cells (hMSC) in vitro can be improved by refined use of experimental parameters. Methods: hMSCs were transduced by adenoviral vectors encoding Luciferase or BMP-2 at a selection of multiplicities of infection (MOI) and exposure times. Transgene production and total protein content were measured. To determine practical relevance, expression of the bone marker genes Runx2 and Type I collagen was analyzed by quantitative PCR. As a phenotypic marker alkaline phosphatase was assessed. ANOVA and post hoc statistical analyses were used to determine differences among data (p < 0.05). Results: Prolonged exposure led to a decrease in transgene production and total protein content. Increasing MOI at exposure of up to 4 hours resulted in a higher production of the transgene. Transfer of the hBMP-2 gene promoted an enhanced lineage progression to the osteoblast phenotype indicating biological activity. Conclusion: Time of exposure is of major importance for toxicity in vitro and should not exceed 4 hours for hMSC. While increase in exposure time leads to cell death, surviving cells, up to a certain limit, seem to compensate by increasing production of the transgene indicating that transduction efficiency cannot be positively measured in a binary yes-or-no scheme.

Schlüsselwörter

humane mesenchymale Stammzellen - adenovirale Transduktion - BMP-2 - osteoblastäre Differenzierung - quantitative real-time PCR

Key words

Human mesenchymal stem cells - adenoviral transduction - BMP-2 - osteoblastic differentiation - quantitative real-time PCR

Literatur

  • 1 Cheng S L, Lou J, Wright N M, Lai C F, Avioli L V, Riew K D. In vitro and in vivo induction of bone formation using a recombinant adenoviral vector carrying the human BMP-2 gene.  Calcif Tissue Int. 2001;  68 87-94
    PubMedGoogle Scholar
  • 2 Lou J, Xu F, Merkel K, Manske P. Gene therapy: adenovirus-mediated human bone morphogenetic protein-2 gene transfer induces mesenchymal progenitor cell proliferation and differentiation in vitro and bone formation in vivo.  J Orthop Res. 1999;  17 43-50
    CrossrefPubMedGoogle Scholar
  • 3 Partridge K, Yang X, Clarke N M, Okubo Y, Bessho K, Sebald W, Howdle S M, Shakesheff K M, Oreffo R O. Adenoviral BMP-2 gene transfer in mesenchymal stem cells: in vitro and in vivo bone formation on biodegradable polymer scaffolds.  Biochem Biophys Res Commun. 2002;  292 144-152
    CrossrefPubMedGoogle Scholar
  • 4 Riew K D, Wright N M, Cheng S, Avioli L V, Lou J. Induction of bone formation using a recombinant adenoviral vector carrying the human BMP-2 gene in a rabbit spinal fusion model.  Calcif Tissue Int. 1998;  63 357-360
    CrossrefPubMedGoogle Scholar
  • 5 Turgeman G, Pittman D D, Muller R, Kurkalli B G, Zhou S, Pelled G, Peyser A, Zilberman Y, Moutsatsos I K, Gazit D. Engineered human mesenchymal stem cells: a novel platform for skeletal cell mediated gene therapy.  J Gene Med. 2001;  3 240-251
    CrossrefPubMedGoogle Scholar
  • 6 Smith P, Shuler F D, Georgescu H I, Ghivizzani S C, Johnstone B, Niyibizi C, Robbins P D, Evans C H. Genetic enhancement of matrix synthesis by articular chondrocytes: comparison of different growth factor genes in the presence and absence of interleukin-1.  Arthritis Rheum. 2000;  43 1156-1164
    CrossrefPubMedGoogle Scholar
  • 7 Niyibizi C, Baltzer A, Lattermann C, Oyama M, Whalen J D, Robbins P D, Evans C H. Potential role for gene therapy in the enhancement of fracture healing.  Clin Orthop. 1998;  335 Suppl S148-S153
    PubMedGoogle Scholar
  • 8 Baltzer A W, Lattermann C, Whalen J D, Wooley P, Weiss K, Grimm M, Ghivizzani S C, Robbins P D, Evans C H. Genetic enhancement of fracture repair: healing of an experimental segmental defect by adenoviral transfer of the BMP-2 gene.  Gene Ther. 2000;  7 734-739
    CrossrefPubMedGoogle Scholar
  • 9 Peterson B, Iglesias R, Zhang J, Wang J C, Lieberman J R. Genetically modified human derived bone marrow cells for posterolateral lumbar spine fusion in athymic rats: beyond conventional autologous bone grafting.  Spine. 2005;  30 283-289
    CrossrefPubMedGoogle Scholar
  • 10 Peterson B, Zhang J, Iglesias R, Kabo M, Hedrick M, Benhaim P, Lieberman J R. Healing of critically sized femoral defects, using genetically modified mesenchymal stem cells from human adipose tissue.  Tissue Eng. 2005;  11 120-129
    CrossrefPubMedGoogle Scholar
  • 11 Sugiyama O, Orimo H, Suzuki S, Yamashita K, Ito H, Shimada T. Bone formation following transplantation of genetically modified primary bone marrow stromal cells.  J Orthop Res. 2003;  21 630-637
    CrossrefPubMedGoogle Scholar
  • 12 Riew K D, Lou J, Wright N M, Cheng S L, Bae K T, Avioli L V. Thoracoscopic intradiscal spine fusion using a minimally invasive gene-therapy technique.  J Bone Joint Surg [Am]. 2003;  85 866-871
    PubMedGoogle Scholar
  • 13 Prockop D J. Marrow stromal cells as stem cells for nonhematopoietic tissues.  Science. 1997;  276 71-74
    CrossrefPubMedGoogle Scholar
  • 14 Pittenger M F, Mackay A M, Beck S C, Jaiswal R K, Douglas R, Mosca J D, Moorman M A, Simonetti D W, Craig S, Marshak D R. Multilineage potential of adult human mesenchymal stem cells.  Science. 1999;  284 143-147
    CrossrefPubMedGoogle Scholar
  • 15 Grove J E, Bruscia E, Krause D S. Plasticity of bone marrow-derived stem cells.  Stem Cells. 2004;  22 487-500
    CrossrefPubMedGoogle Scholar
  • 16 Musgrave D S, Bosch P, Lee J Y, Pelinkovic D, Ghivizzani S C, Whalen J, Niyibizi C, Huard J. Ex vivo gene therapy to produce bone using different cell types.  Clin Orthop. 2000;  378 290-305
    PubMedGoogle Scholar
  • 17 Niyibizi C, Smith P, Mi Z, Phillips C L, Robbins P. Transfer of proalpha2(I) cDNA into cells of a murine model of human Osteogenesis Imperfecta restores synthesis of type I collagen comprised of alpha1(I) and alpha2(I) heterotrimers in vitro and in vivo.  J Cell Biochem. 2001;  83 84-91
    CrossrefPubMedGoogle Scholar
  • 18 AppliedBiosystems .User Bulletin #2: ABI Prism 7700 Sequence Detection System. AppliedBiosystems, Foster City, CA updated 10/2001
    Google Scholar
  • 19 Lehrman S. Virus treatment questioned after gene therapy death.  Nature. 1999;  401 517-518
    PubMedGoogle Scholar
  • 20 Kaiser J. Gene therapy. Seeking the cause of induced leukemias in X-SCID trial.  Science. 2003;  299 495
    CrossrefPubMedGoogle Scholar
  • 21 Check E. A tragic setback.  Nature. 2002;  420 116-118
    PubMedGoogle Scholar
  • 22 Van Damme A, Vanden Driessche T, Collen D, Chuah M K. Bone marrow stromal cells as targets for gene therapy.  Curr Gene Ther. 2002;  2 195-209
    PubMedGoogle Scholar
  • 23 Studeny M, Marini F C, Dembinski J L, Zompetta C, Cabreira-Hansen M, Bekele B N, Champlin R E, Andreeff M. Mesenchymal stem cells: potential precursors for tumor stroma and targeted-delivery vehicles for anticancer agents.  J Natl Cancer Inst. 2004;  96 1593-1603
    CrossrefPubMedGoogle Scholar
  • 24 Kurozumi K, Nakamura K, Tamiya T, Kawano Y, Ishii K, Kobune M, Hirai S, Uchida H, Sasaki K, Ito Y, Kato K, Honmou O, Houkin K, Date I, Hamada H. Mesenchymal stem cells that produce neurotrophic factors reduce ischemic damage in the rat middle cerebral artery occlusion model.  Mol Ther. 2005;  11 96-104
    PubMedGoogle Scholar
  • 25 Chang S C, Chuang H L, Chen Y R, Chen J K, Chung H Y, Lu Y L, Lin H Y, Tai C L, Lou J. Ex vivo gene therapy in autologous bone marrow stromal stem cells for tissue-engineered maxillofacial bone regeneration.  Gene Ther. 2003;  10 2013-2019
    CrossrefPubMedGoogle Scholar
  • 26 Baltzer A W, Whalen J D, Stefanovic-Racic M, Ziran B, Robbins P D, Evans C H. Adenoviral transduction of human osteoblastic cell cultures: a new perspective for gene therapy of bone diseases.  Acta Orthop Scand. 1999;  70 419-424
    PubMedGoogle Scholar
  • 27 Ducy P, Zhang R, Geoffroy V, Ridall A L, Karsenty G. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation.  Cell. 1997;  89 747-754
    CrossrefPubMedGoogle Scholar

OA Dr. med. H. Koch

Klinik für Orthopädie und orthopädische Chirurgie der Ernst-Moritz-Arndt-Universität

Sauerbruchstraße

17487 Greifswald

Phone: 0 38 34/86 72 27

Fax: 0 38 34/86 72 22

Email: hannjoerg.koch@uni-greifswald.de

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