Search

Human mesenchymal stem cells (hMSCs) are under research for applications in cell-based therapies due to hMSCs therapeutic properties. However, the therapeutic properties of hMSCs could be advanced by genetic modification. Viral modification of hMSCs is highly efficient but presents safety and production issues that limits clinical translation. Conversely, nonviral gene delivery offers advantages in safety and production compared to viral approaches, but is less efficient, especially in hMSCs. To address the challenges of nonviral gene delivery in hMSCs, our group has shown that priming hMSCs with the glucocorticoid dexamethasone can significantly increase transfection compared to a vehicle control (VC). Work presented in this dissertation expands our priming library for hMSC transfection by screening clinically approved drugs for compounds that significantly modulate transfection compared to the VC. Our results indicate that glucocorticoids are a potent priming drug class in hMSCs, but other compounds, such as antibiotics and antihypertensives, can also modulate transfection, potentially through unique molecular mechanisms. The molecular mechanisms of the identified priming compounds were further investigated using Drug Set Enrichment Analysis to predict endogenous genes that might be modulated by hMSC transfection priming. Our results indicate that endogenous genes related to Wnt and interferon signaling are modulated by antibiotics and antihypertensives, providing new molecular targets for future priming studies. In addition to priming, work presented in this dissertation systematically compared key transfection parameters to optimize transfection in hMSCs. Our results motivate the use of minimized DNA vectors for hMSC transfection, and suggest the need for optimization of transfection parameters on a donor and tissue source basis as these two parameters can create large variation in transfection outcomes. Finally, we show that efficient expression of multiple tra

Nonviral gene delivery, though limited by inefficiency, has extensive utility in cell therapy, tissue engineering, and diagnostics. Substrate-mediated gene delivery (SMD) increases efficiency and allows transfection at a cell-biomaterial interface, by immobilizing and concentrating nucleic acid complexes on a surface. Efficient SMD generally requires substrates to be coated with serum or other protein coatings to mediate nucleic acid complex immobilization, as well as cell adhesion and growth; however, this strategy limits reproducibility and may be difficult to translate for clinical applications. As an alternative, we screened a chemically defined combinatorial library of 20 different extracellular matrix mimetic substrates containing combinations of (1) different sulfated polysaccharides that are essential extracellular matrix glycosaminoglycans (GAGs), with (2) mimetic peptides derived from adhesion proteins, growth factors, and cell-penetrating domains, for use as SMD coatings. We identified optimal substrates for DNA lipoplex and polyplex SMD transfection of fibroblasts and human mesenchymal stem cells. Optimal extracellular matrix mimetic substrates varied between cell type, donor source, and transfection reagent, but typically contained Heparin GAG and an adhesion peptide. Multiple substrates significantly increased transgene expression (i.e. 2- to 20-fold) over standard protein coatings. Considering previous research of similar ligands, we hypothesize extracellular matrix mimetic substrates modulate cell adhesion, proliferation, and survival, as well as plasmid internalization and trafficking. Our results demonstrate the utility of screening combinatorial extracellular matrix mimetic substrates for optimal SMD transfection towards application- and patient-specific technologies.

Substrate mediated gene delivery (SMD) is a method of immobilizing DNA complexes to a substrate via covalent attachment or nonspecific adsorption, which allows for increased transgene expression with less DNA compared to traditional bolus delivery. It may also increase cells receptivity to transfection via cell-material interactions. Substrate modifications with poly(acrylic) acid (PM) brushes may improve SMD by enhancing substrate interactions with DNA complexes via tailored surface chemistry and increasing cellular adhesion via moieties covalently bound to the brushes. Previously, we described a simple method to graft PM brushes to Ti and further demonstrated conjugation of cell adhesion peptides (i.e., RGD) to the PM brushes to improve biocompatibility. The objective of this work was to investigate the ability of Ti substrates modified with PM-RGD brushes (PM-RGD) to immobilize complexes composed of branched polyethyleneimine and DNA plasmids (bPEI-DNA) and support SMD in NIH/3T3 fibroblasts. Transfection in NIH/3T3 cells cultured on bPEI-DNA complexes immobilized onto PM-RGD substrates was measured and compared to transfection in cells cultured on control surfaces with immobilized complexes including Flat Ti, PM brushes modified with a control peptide (RGE), and unmodified PM. Transfection was two-fold higher in cells cultured on PM-RGD compared to those cultured on all control substrates. While DNA immobilization measured with radiolabeled DNA indicated that all substrates (PM-RGD, unmodified PM, Flat Ti) contained nearly equivalent amounts of loaded DNA, ellipsometric measurements showed that more total mass (i.e., DNA and bPEI, both complexed and free) was immobilized to PM and PM-RGD compared to Flat Ti. The increase in adsorbed mass may be attributed to free bPEI, which has been shown to improve transfection. Further transfection investigations showed that removing free bPEI from the immobilized complexes decreased SMD transfection and negated any differenc, Funding Agencies|Nebraska Research Initiative,; University of Nebraska-Lincoln Tobacco Settlement Funds; National Institutes of Health [1 DP2 EB025760-01]; Leibniz Institute of Polymer Research Dresden; National Science Foundation (CAREER) [CBET1254415]; Center for Nanohybrid Functional Materials [NSF EPS-1004094]; J.A. Woollam Company; USDA CSREES-Nebraska [NEB-21-146]