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Both outcomes varied widely as a function of each parameter, e.g., D3 bone-marrow-derived hMSCs (hBMSCs) transfected with the conventional CMV plasmid complexed with Turbofect produced transfection efficiencies around 35%, the highest of our study ( Figure 2A), while D2 adipose-derived hMSCs (hAMSCs) transfected with CMV MIP complexed with Lipofectamine 3000 produced the highest transgenic luciferase activity of our study ( Figure 2D). Given all of the variables above, this study tested 64 conditions, in triplicate on duplicate days (n = 6), to identify key transfection parameters ( Tables 1 Table S1) that modulate hMSC transfection efficiency ( Figures 2A and 2C ) and transgene production ( Figures 2B and 2D). It is important to note that due to the varying sizes of DNA vectors and promoters tested, both mass of DNA delivered and DNA copy number (i.e., molarity of transgene) were normalized for each promoter and cationic carrier in order to properly compare conditions ( Table 2). Imaging results were then verified by a chemical assay for transgenic luciferase activity, in relative light units (RLUs), normalized by total cellular protein values (RLU/mg Protein). The effects on transfection efficiency were assayed by fluorescence imaging of an expressed transgenic fusion protein of enhanced green fluorescent protein (EGFP) with luciferase, normalized by total cell count (Hoechst 33342, nuclei stain), to obtain transfection efficiencies for all conditions. Specifically, we investigated two promoters (CMV and EF1a Table 1) and four DNA vectors (plasmid, plasmid with no F1 origin of replication, MC, and MIP Table 1 Figure 1), delivered using two commercially available cationic carriers (Turbofect and Lipofectamine 3000 ) to cells from four hMSC donors (D1, D2, D3, and D4 Table S1) derived from two tissue sources (bone-marrow-derived and adipose-derived Table S1) in order to identify transfection parameters that significantly (p < 0.05) affect transfection efficiency and transgene production in hMSCs.
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The objective of this study was to investigate the effects of different promoters, DNA vectors, and cationic carriers on transfection in hMSCs from different donors and tissues.
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The data presented in this work can guide the development of other hMSC transfection systems with the goal of producing clinically relevant, genetically modified hMSCs. Following systematic comparison of each variable, we identified adipose-derived hMSCs transfected with mini-intronic plasmids containing the CMV promoter delivered using Lipofectamine 3000 as the parameters that produced the highest transfection levels. Specifically, we investigated two promoters (cytomegalovirus and elongation factor 1 alpha), four DNA vectors (plasmid, plasmid with no F1 origin, minicircle, and mini-intronic plasmid), two cationic carriers (Lipofectamine 3000 and Turbofect), and four donors of hMSCs from two tissues (adipose and bone marrow) for efficient hMSC transfection. This work takes a complementary approach to addressing the challenges of transfecting hMSCs by systematically investigating key transfection parameters for their effect on transgene expression. However, even with priming, hMSC transfection remains inefficient for clinical applications. To address the shortcomings of nonviral gene delivery to hMSCs, our lab has previously demonstrated that pharmacological “priming” of hMSCs with clinically approved drugs can increase transfection in hMSCs by modulating transfection-induced cytotoxicity. However, gene delivery, particularly through nonviral routes, is inefficient. Human mesenchymal stem cells (hMSCs) are primary cells with high clinical relevance that could be enhanced through genetic modification. Gene Editing: Technology & Applications.