Mechanisms of unprimed and dexamethasone‐primed nonviral gene delivery to human mesenchymal stem cells

Abstract

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 functional expression of therapeutic transgenes or control over behavior and differentiation. Viral gene delivery is efficient, but suffers from safety issues, while nonviral methods are safe, but highly inefficient, especially in hMSCs. Our lab previously demonstrated that priming cells before delivery of DNA complexes with dexamethasone (DEX), an anti‐inflammatory glucocorticoid drug, significantly increases hMSC transfection success. This work systematically investigates the mechanisms of hMSC transfection and DEX‐mediated enhancement of transfection. Our results show that hMSC transfection and its enhancement by DEX are decreased by inhibiting classical intracellular transport and nuclear import pathways, but DEX transfection priming does not increase cellular or nuclear internalization of plasmid DNA (pDNA). We also show that hMSC transgene expression is largely affected by pDNA promoter and enhancer sequence changes, but DEX‐mediated enhancement of transfection is unaffected by any pDNA sequence changes. Furthermore, DEX‐mediated transfection enhancement is not the result of increased transgene messenger RNA transcription or stability. However, DEX‐priming increases total protein synthesis by preventing hMSC apoptosis induced by transfection, resulting in increased translation of transgenic protein. DEX may also promote further enhancement of transgenic reporter enzyme activity by other downstream mechanisms. Mechanistic studies of nonviral gene delivery will inform future rationally designed technologies for safe and efficient genetic modification of clinically relevant cell types.

High-throughput screening of clinically approved drugs that prime nonviral gene delivery to human Mesenchymal stem cells

“…An efficient nonviral gene delivery system for ex vivo genetic modification of clinically relevant hMSCs is lacking, however, our lab has previously demonstrated that pharmacological priming, or the addition of compounds to the culture media to modulate the cellular response to transfection, is a simple and effective method to enhance transfection in multiple cell types [15][16][17][23][24][25][26]. The idea of priming was developed after our studies using microarray analysis of transfected versus treated, but untransfected cells, where differentially expressed endogenous genes were identified between each condition in HEK 293 T cells and the expression of these identified genes were perturbed pharmacologically (i.e.…”

“…An alternative approach to improving gene delivery to hMSCs is the idea of priming, where a compound is added to cultured cells to modulate the cellular response to nonviral gene delivery. Our lab has demonstrated that an antiinflammatory glucocorticoid drug, dexamethasone, can prime transfection to hMSCs from multiple donors and tissue sources by simple supplementation to the culture media 0-30 min prior to transfection [15,16]. With dexamethasone priming, we showed an increase in transgenic luciferase activity by 10-to 15-fold and an increase in the proportion of hMSCs expressing transgenic enhanced green fluorescent protein (EGFP) (i.e.…”

“…transfection efficiency) by about three-fold, relative to a vehicle control (VC) [15]. We elucidated that dexamethasone enhances nonviral gene delivery to hMCSs by reducing transfection-induced metabolic decline [15], rescuing transfectioninduced protein synthesis inhibition [16], and preventing apoptosis [16], all while retaining differentiation capacity of the cells [15].…”

“…Our previous work has established chemical priming as a simple method to enhance gene delivery to hMSCs, however, few compounds have been identified that can prime nonviral gene delivery to hMSCs [15][16][17][18][19][20], and the underlying biological processes that are necessary for successful transfection are not well understood. To address these issues, a drug repurposing approach similar to our previous screen for compounds that prime polymer-mediated gene delivery to human embryonic kidney cells (HEK 293 T) [17] was used in this current work to generate a library of compounds from the National Institutes of Health Clinical Collection (NCC), a collection of over 700 clinically approved drugs, belonging to diverse drug classes, made available for drug repurposing [21], which prime lipid-mediated nonviral gene delivery to adipose-derived hMSCs (hAMSCs) from two human donors at four concentrations spanning four orders of magnitude.…”

Influence of anti‐allergic drugs used systemically on the process of root resorption during delayed tooth replantation: A study in rats

“…Glucocorticosteroids are an efficient and widely used treatment for allergic respiratory diseases and are therefore considered anti‐allergic drugs . Low doses can stimulate the production of OPG and cause disassembly of cytoskeletal actin fibers present in clear and sealing zones of osteoclasts, which may interfere with the resorption process . Quercetin, a natural flavonoid, inhibits the release of histamine and leukotrienes and is also considered an anti‐allergic drug .…”