Comparison and Optimization of Novel Nonviral Vectors for Gene Transfer in Human Bone Marrow-Derived Mesenchymal Stem Cells

METHODS: The plasmid expression vectors employed included pCMVLuc carrying the Firefly luciferase gene and pCMVhIGF-I carrying a human insulin-like growth factor I (hIGF-I) cDNA as control treatment [1-3]. Bone marrow aspirates were obtained from the femurs of normal donors during knee endoprothesis and MSCs were isolated and expanded in culture using standard protocols [4-6]. Briefly, the aspirates were layered onto Histopaque-1077 density gradient (Sigma), centrifuged, and the nucleated cell fraction at the interface was collected, washed, and resuspended in human MSC (hMSC) growth medium (MesenCult® MSC Basal Human; StemCell Technologies Inc.) containing MSC Stimulatory Supplements (StemCell Technologies Inc.). The cells were characterized for stem cell surface markers and multilineage potential (CD44, CD31, CD34, CD71, and CD105) [4]. Prior to transfection, fresh growth medium was added to the cultures. Cells (passage 1-2) were transfected in 96-well plates (5 x 10/well) at 60-70% confluence according to the manufacturer’s instructions using FuGENE 6 (Fg; Roche Applied Sciences; 0.25 μg DNA/0.5 μl reagent), Metafectene (Mf; Biontex; 0.125 μg DNA/0.625 μl reagent), Lipofectamine 2000 (Lp; Invitrogen; 0.2 μg DNA/0.5 μl reagent), Dreamfect (Df; Oz Bioscience; 0.125 μg DNA/0.5 μl reagent), Gene Jammer (GJ; Stratagene; 0.25 μg DNA/0.75 μl reagent), Effectene (Ef; Qiagen; 0.05 μg DNA/1.25 μl reagent), Turbofectin (Tf; OriGene; 0.05 μg DNA/0.15 μl reagent), Mirus (Ms; Mirus Bio Corporation; 0.1 μg DNA/0.28 μl reagent), Lipofectamin with PLUS (Lp+P; Invitrogen; 0.1 μg DNA/0.3 μl reagent), Gene Juice (GJu; Novagen; 0.06 μg DNA/0.2 μl reagent), Dreamfect Gold (DfG; Oz Bioscience; 0.25 μg DNA/1 μl reagent), Transpass D2 (TP; New England Biolabs; 0.1 μg DNA/0.2 μl reagent), Jet Pei (JP; Polyplus Transfection; 0.25 μg DNA/0.5 μl reagent), Ecotransfect (Ec; Oz Bioscience; 0.2 μg DNA/0.4 μl reagent), DMRIEC (DC; Invitrogen; 0.06 μg DNA/0.18 μl reagent) [1,7]. In addition to the standard protocols, hyaluronidase (4 U/ml) (Sigma) was added before and during transfection in each set of experiments [1]. Two days after transfection, transgene expression was determined using a luciferase assay (Promega). The amounts of total proteins were measured using the BCA kit (Thermo Scientific). Transfection efficiencies were determined as relative light unit (RLU) per mg of total proteins. Evaluation of the cytotoxicity in transfected cells was performed using the Cytotoxicity Detection Kit (LDH) (Roche Applied Sciences) by measuring the release of lactate dehydrogenase activity from damaged cells. Data are given as % cytotoxicity [(exp. value low control/high control low control) x100] where ”low control“ corresponds to untransfected cells and ”high control“ to cells placed in the lysis buffer provided in the kit. Each condition was performed in duplicate in 3 independent experiments. Data are expressed as mean ± SD. The Mann-Whitney Rank Sum Test was employed where appropriate with P < 0.05 considered statistically significant.