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Efficient plasmid-mediated gene transfection of ovine bone marrow mesenchymal stromal cells.
MedLine Citation:
PMID:  23321328     Owner:  NLM     Status:  In-Data-Review    
Abstract/OtherAbstract:
BACKGROUND AIMS: Given the close similarity between ovine and human cardiomyocytes, sheep models of myocardial infarction and heart failure are increasingly used in studies of stem cell-mediated heart regeneration. In these studies, mesenchymal stromal cells (MSCs) are frequently employed. To enhance the paracrine effects of these MSCs, ex vivo transfection with genes encoding growth factors has been proposed. Although viral vectors exhibit higher transfection efficiency than plasmids, they entail the risks of uncontrolled transgene expression and immune reactions that preclude repeated administration. Our aim was to optimize the efficiency of plasmid-mediated transfection of ovine MSCs, while preserving cell viability.
METHODS: Varying amounts of diverse cationic lipids were used to obtain the reagent-to-DNA mass ratio showing highest luciferase activity. Transfection efficiency (flow cytometry) was tested on plasmid-green fluorescent protein-transfected MSCs at increasing DNA mass.
RESULTS: Lipofectamine LTX 5 μL and Plus reagent 4 μL with 2 μg of DNA yielded 42.3 ± 4.7% transfection efficiency, while preserving cell viability. Using these transfection conditions, we transfected MSCs with a plasmid encoding human vascular endothelial growth factor (VEGF) and found high VEGF protein concentrations in the culture supernatant from day 2 (1968 ± 324 pg/mL per μg DNA) through at least day 12 (888 ± 386 pg/mL per μg DNA) after transfection.
CONCLUSIONS: Plasmid-mediated transfection of ovine MSCs to over-express paracrine heart-regenerative growth factors is feasible and efficient and overcomes the risks and limitations associated with the use of viral vectors.
Authors:
Paola Locatelli; Fernanda Daniela Olea; Anna Hnatiuk; Diana Sepúlveda; Juan Manuel Pérez Sáez; Rafael Argüello; Alberto Crottogini
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Cytotherapy     Volume:  15     ISSN:  1477-2566     ISO Abbreviation:  Cytotherapy     Publication Date:  2013 Feb 
Date Detail:
Created Date:  2013-01-16     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  100895309     Medline TA:  Cytotherapy     Country:  England    
Other Details:
Languages:  eng     Pagination:  163-70     Citation Subset:  IM    
Copyright Information:
Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.
Affiliation:
Department of Physiology, Favaloro University, Buenos Aires, Argentina.
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