Objective: To define the potential of endothelial progenitor cells (EPCs) to internalize and express exogenous DNA by nonviral transfection. Liposome-mediated delivery of either non-coding (FITC-DNA) or coding (pEGFP C1) DNA sequences was compared in view of their potential use for therapeutic strategies. Methods: Peripheral blood mononuclear cells (PBMNCs) were isolated from healthy volunteers. 1X106/cm2 PBMNCs were plated in EGM2-MV on fibronectin-coated flasks. After 6 days of culture, adherent cells were incubated with DiI-AcLDL and FITC-UEA-I. Double-positive cells were identified as EPCs. EPCs were detached after 6 days of culture and seeded at 8X105 cells/cm2 the day before the transfection. Different cationic liposomes were used for the transfection: Ap41 (with FITC-DNA); PolyFect, Effectene and Superfect with plasmid DNA. DNA was added to liposomes and the mixture was incubated for 15 minutes at room temperature. EPCs were exposed to the transfection mixture for 4 hours (FITC-DNA) or 24 hours (plasmid-DNA). Thereafter, EPCs were detached and the detection of cells containing FITC-DNA or expressing GFP was performed by FACS. For the detection of FITC-DNA, EPCs were washed with glycine 0.2M pH 2.8 to remove any fluorescent extracellular complex. Results: After 6 days of culture an adherent population with an endothelial-like phenotype (EPCs) was obtained, as confirmed by AcLDL/UEA-I positivity. 82.5% of EPCs internalized FITC-DNA at the end of 4 hours transfection; the number increased to 88.2% 20 hours later. No EPCs expressed GFP, with none of the three transfectants. Moreover, PolyFect and SuperFect seemed to induce the detachment of some adherent cells, because an appreciable amount of floating cells at the end of transfection was observed. Conclusion: While the uptake of the complex FITC-DNA/liposome by EPCs was very efficient, the plasmid gene transfer had no success. This finding may be explained by a lack of genomic integration and a rapid degradation of the plasmid DNA.
Nonviral transfection of endothelial progenitor cells
SANTONI, TATIANA;DI STEFANO, ROSSELLA;BALBARINI, ALBERTO
2005-01-01
Abstract
Objective: To define the potential of endothelial progenitor cells (EPCs) to internalize and express exogenous DNA by nonviral transfection. Liposome-mediated delivery of either non-coding (FITC-DNA) or coding (pEGFP C1) DNA sequences was compared in view of their potential use for therapeutic strategies. Methods: Peripheral blood mononuclear cells (PBMNCs) were isolated from healthy volunteers. 1X106/cm2 PBMNCs were plated in EGM2-MV on fibronectin-coated flasks. After 6 days of culture, adherent cells were incubated with DiI-AcLDL and FITC-UEA-I. Double-positive cells were identified as EPCs. EPCs were detached after 6 days of culture and seeded at 8X105 cells/cm2 the day before the transfection. Different cationic liposomes were used for the transfection: Ap41 (with FITC-DNA); PolyFect, Effectene and Superfect with plasmid DNA. DNA was added to liposomes and the mixture was incubated for 15 minutes at room temperature. EPCs were exposed to the transfection mixture for 4 hours (FITC-DNA) or 24 hours (plasmid-DNA). Thereafter, EPCs were detached and the detection of cells containing FITC-DNA or expressing GFP was performed by FACS. For the detection of FITC-DNA, EPCs were washed with glycine 0.2M pH 2.8 to remove any fluorescent extracellular complex. Results: After 6 days of culture an adherent population with an endothelial-like phenotype (EPCs) was obtained, as confirmed by AcLDL/UEA-I positivity. 82.5% of EPCs internalized FITC-DNA at the end of 4 hours transfection; the number increased to 88.2% 20 hours later. No EPCs expressed GFP, with none of the three transfectants. Moreover, PolyFect and SuperFect seemed to induce the detachment of some adherent cells, because an appreciable amount of floating cells at the end of transfection was observed. Conclusion: While the uptake of the complex FITC-DNA/liposome by EPCs was very efficient, the plasmid gene transfer had no success. This finding may be explained by a lack of genomic integration and a rapid degradation of the plasmid DNA.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
