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Generation of human adult mesenchymal stromal/stem cells expressing defined xenogenic vascular endothelial growth factor levels by optimized transduction and flow cytometry purification

Helmrich, U. and Marsano, A. and Melly, L. and Wolff, T. and Christ, L. and Heberer, M. and Scherberich, A. and Martin, I. and Banfi, A.. (2012) Generation of human adult mesenchymal stromal/stem cells expressing defined xenogenic vascular endothelial growth factor levels by optimized transduction and flow cytometry purification. Tissue engineering. Part C, Methods, Vol. 18, H. 4. pp. 283-292.

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Official URL: http://edoc.unibas.ch/dok/A6031551

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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 VEGF(164) 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.
Faculties and Departments:03 Faculty of Medicine > Departement Biomedizin > Department of Biomedicine, University Hospital Basel > Cell and Gene Therapy (Banfi)
03 Faculty of Medicine > Departement Biomedizin > Department of Biomedicine, University Hospital Basel > Tissue Engineering (Martin)
03 Faculty of Medicine > Bereich Operative Fächer (Klinik) > Innere Organe > Gefässchirurgie (Stierli)
03 Faculty of Medicine > Departement Klinische Forschung > Bereich Operative Fächer (Klinik) > Innere Organe > Gefässchirurgie (Stierli)
03 Faculty of Medicine > Bereich Operative Fächer (Klinik) > Querschnittsbereich Forschung > Chirurgische Forschung (Heberer)
03 Faculty of Medicine > Departement Klinische Forschung > Bereich Operative Fächer (Klinik) > Querschnittsbereich Forschung > Chirurgische Forschung (Heberer)
03 Faculty of Medicine > Departement Biomedizin > Department of Biomedicine, University Hospital Basel > Cardiac Surgery and Engineering (Marsano)
UniBasel Contributors:Helmrich, Uta and Scherberich, Arnaud and Heberer, Michael and Martin, Ivan and Banfi, Andrea and Marsano, Anna
Item Type:Article, refereed
Article Subtype:Research Article
Bibsysno:Link to catalogue
Publisher:Mary Ann Liebert
ISSN:1937-3384
Note:Publication type according to Uni Basel Research Database: Journal article
Language:English
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Last Modified:31 Dec 2015 10:52
Deposited On:26 Apr 2013 06:58

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