Human mesenchymal stem cells (hMSCs) are under research for applications in tissue engineering and cell-based therapies due to hMSCs therapeutic properties. However, the therapeutic properties of hMSCs could be further 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 defense expands our priming library for hMSC transfection by screening 707 clinically approved drugs for compounds that significantly modulate transfection compared to a VC. Our results indicate that glucocorticoids are a potent priming drug class in hMSCs, but other compounds, such as antibiotics and antihypertensives, can also significantly modulate transfection compared to the VC, potentially through unique molecular mechanisms. The molecular mechanisms of the newly identified priming compounds were further investigated using Drug Set Enrichment Analysis to predict specific endogenous genes that might be modulated by hMSC transfection priming. Our results indicate that specific 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 defense systematically compared key transfection parameters to optimize transfection in hMSCs. Our results motivate the use of minimized DNA vectors for hMSC transfection, as well as 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 transgenes simultaneously in hMSCs can be achieved using a poly-cistronic DNA vector with a dual 2A peptide sequence. These studies inform methods to use nonviral gene delivery to advance hMSC clinical applications, with emphasis on treatment of multifaceted diseases that require expression of multiple transgenes.