eNOS-transfected endothelial progenitor cells (Northern Therapeutics, Inc.)

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.