Generation of Human Adult Mesenchymal Stromal/Stem Cells Expressing Defined Xenogenic Vascular Endothelial Growth Factor Levels by Optimized Transduction and Flow Cytometry Purification
- 1 April 2012
- journal article
- research article
- Published by Mary Ann Liebert Inc in Tissue Engineering, Part C: Methods
- Vol. 18 (4), 283-292
- https://doi.org/10.1089/ten.tec.2011.0413
Abstract
Adult mesenchymal stromal/stem cells (MSCs) are a valuable source of multipotent progenitors for tissue engineering and regenerative medicine, but may require to be genetically modified to widen their efficacy in therapeutic applications. For example, overexpression of the angiogenic factor vascular endothelial growth factor (VEGF) at controlled levels is an attractive strategy to overcome the crucial bottleneck of graft vascularization and to avoid aberrant vascular growth. Since the regenerative potential of MSCs is rapidly lost during in vitro expansion, we sought to develop an optimized technique to achieve high-efficiency retroviral vector transduction of MSCs derived from both adipose tissue (adipose stromal cells, ASCs) or bone marrow (BMSCs) and rapidly select cells expressing desired levels of VEGF with minimal in vitro expansion. The proliferative peak of freshly isolated human ASCs and BMSCs was reached 4 and 6 days after plating, respectively. By performing retroviral vector transduction at this time point, >90% efficiency was routinely achieved before the first passage. MSCs were transduced with vectors expressing rat VEGF164 quantitatively linked to a syngenic cell surface marker (truncated rat CD8). Retroviral transduction and VEGF expression did not affect MSC phenotype nor impair their in vitro proliferation and differentiation potential. Transgene expression was also maintained during in vitro differentiation. Furthermore, three subpopulations of transduced BMSCs homogeneously producing specific low, medium, and high VEGF doses could be prospectively isolated by flow cytometry based on the intensity of their CD8 expression already at the first passage. In conclusion, this optimized platform allowed the generation of populations of genetically modified MSCs, expressing specific levels of a therapeutic transgene, already at the first passage, thereby minimizing in vitro expansion and loss of regenerative potential.Keywords
This publication has 39 references indexed in Scilit:
- Paracrine factors released by GATA-4 overexpressed mesenchymal stem cells increase angiogenesis and cell survivalAmerican Journal of Physiology-Heart and Circulatory Physiology, 2010
- Adipose tissue‐derived progenitors for engineering osteogenic and vasculogenic graftsJournal of Cellular Physiology, 2010
- High-Throughput Flow Cytometry Purification of Transduced Progenitors Expressing Defined Levels of Vascular Endothelial Growth Factor Induces Controlled Angiogenesis In VivoThe International Journal of Cell Cloning, 2009
- Mesenchymal Stromal Cells: Current Understanding and Clinical StatusThe International Journal of Cell Cloning, 2009
- Telomerase Immortalized Human Amnion- and Adipose-Derived Mesenchymal Stem Cells: Maintenance of Differentiation and Immunomodulatory CharacteristicsTissue Engineering, Part A, 2009
- A Perivascular Origin for Mesenchymal Stem Cells in Multiple Human OrgansCell Stem Cell, 2008
- Stem Cells from Adipose Tissue Allow Challenging New Concepts for Regenerative MedicineTissue Engineering, 2007
- Mesenchymal stem cells overexpressing Akt dramatically repair infarcted myocardium and improve cardiac function despite infrequent cellular fusion or differentiationMolecular Therapy, 2006
- Microenvironmental VEGF distribution is critical for stable and functional vessel growth in ischemiaThe FASEB Journal, 2006
- Real‐time quantitative RT‐PCR analysis of human bone marrow stromal cells during osteogenic differentiation in vitroJournal of Cellular Biochemistry, 2002