Transfection optimization of ovine bone marrow (BM) mesenchymal stromal cells (MSCs) for myocardial regeneration

LOCATELLI P, OLEA FD, PÉREZ SÁEZ JM, SEPÚLVEDA D, HNATIUK A, BUSSMANN UA, BERCOVICH A, CROTTOGINI A.

La Plata, Argentina

Congreso; XVII Annual Meeting of the International Society for Heart Research Latin American Section; 2010

Interantional Society for Heart Research Latin American Section

MSCs have shown benefit in myocardial infarction (MI). Besides, VEGF gene transfer induces angiogenesis and cardiomyogenesis in ovine MI. Therefore MSCs overexpressing VEGF would potentiate their benefits. We aimed to identify and replicate ovine BM MSCs and optimize transfection efficiency (TE). BM was aspirated from the iliac crest. Plastic-adherent cells were cultured until p4. MSCs were identified by their CD44+, CD166+ and CD45− phenotype (flow citometry) and differentiation into adipocytes, condrocytes and osteocytes. TE was optimized by testing different cationic lipids to DNA ratios (GeneJuice, Lipofectamine 2000 and LTX; and 1-6 µg GFP or luciferase reporter plasmids). Once optimal TE was established (using luciferase activity assessments), MSCs were transfected with a plasmid encoding hVEGF165, marked with CMDiI and injected in the peri-infarct of 4 sheep with MI. Results: TE was optimal (maximal luciferase activity) with 2,5 µl lipofectamine LTX and 2 µl Plus reagent/µg DNA/cm2 (corresponding to 6.5% GFP+ by flow citometry). VEGF-transfected MSCs were detected in myocardium at 6 and 30 days post-implant. Conclusion: Ovine BM MSCs are identifiable and replicable. Transfected cells persist viable for at least 30 days post-implant, indicating their usefulness in heart regeneration research.