Dr. Angela K. Pannier
Date of this Version
Hamann A. Enhancing Non-Viral Gene Delivery to Human Mesenchymal Stem Cells Using Glucocorticoid Priming. M.S. thesis, University of Nebraska-Lincoln. 2017
Human mesenchymal stem cells (hMSCs) are under intense study for applications of cell and gene therapeutics because of their unique immunomodulatory and regenerative properties. Safe and efficient genetic modification of hMSCs could increase their clinical potential by allowing expression of therapeutic transgenes and control over behavior and differentiation. Viral gene delivery methods are efficient, but suffer from safety issues, while non-viral methods are safe, but are highly inefficient, especially in hMSCs. Our lab previously demonstrated that priming hMSCs with dexamethasone (DEX), a glucocorticoid drug, significantly increases transfection success. This thesis investigates the mechanisms of DEX-mediated enhancement. Our results show that DEX priming of transfection is mediated by binding of the cytosolic glucocorticoid receptor (GR) and can be increased by overexpression of GR. We also show that the importin β-mediated nuclear import pathway is important to hMSC transfection, but DEX acts independent of classical nuclear import pathways and does not increase nuclear internalization of pDNA, but does increase cellular internalization of pDNA. We also show that DEX enhancement of transfection occurs regardless of pDNA sequence element changes. In addition, we expand DEX priming to hMSCs derived from several different tissues and human donors, and demonstrate significant variability of transfection and responsiveness to DEX priming between tissue and donor source, reiterating the concern of variability in clinically relevant cell types. We also show DEX priming effects are cell-type dependent in several other cell lines. Mechanistic study of non-viral gene delivery and DEX priming will inform future rationally designed technologies for safe and efficient transfection of clinically relevant cell types.
Advisor: Angela K. Pannier