Bone marrow-derived mononuclear cells are isolated and differentiated into an endothelial lineage (EPC), and then transfected with the therapeutic gene (heNOS).
Mononuclear cells (MNCs) are isolated by density gradient centrifugation at 400g for 30 minutes. BM-MNCs are resuspended in differential endothelial cell culture medium (EBM-2) with FBS (10%), penicillin (50 U/mL), streptomycin (50 μg/mL), and L-glutamine (2 mmol/L), plated on gelatin-coated tissue culture flasks and incubated at 37°C with 5% CO2 for 7-10 days, to produce endothelial-like progenitor cells (ELPCs). ELPCs are transfected, via electroporation (MaxCyte), with human eNOS cloned into the pVax-1 plasmid vector, according to a protocol optimized by the manufacturer. After transfection, cells are replated and cultured for 24 hours before injection.