title: Efficient plasmid-mediated gene transfection of ovine bone marrow mesenchymal stromal cells.
abstract: OBJECTIVE
  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: Department of Physiology, Favaloro University, Buenos Aires, Argentina.
authors: Paola
authors: P
authors: Locatelli
authors: Fernanda Daniela
authors: FD
authors: Olea
authors: Anna
authors: A
authors: Hnatiuk
authors: Diana
authors: D
authors: Sepúlveda
authors: Juan Manuel
authors: JM
authors: Pérez Sáez
authors: Rafael
authors: R
authors: Argüello
authors: Alberto
authors: A
authors: Crottogini
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D064987:Cell- and Tissue-Based Therapy; D015316:Genetic Therapy; D006321:Heart / Q000502:physiology; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D032383:Myocytes, Cardiac / Q000378:metabolism; D019899:Paracrine Communication; D010957:Plasmids; D012038:Regeneration; D034561:Sheep, Domestic / Q000235:genetics*; D014162:Transfection / Q000379:methods; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000378:metabolism
chemical_list: D042461:Vascular Endothelial Growth Factor A