Glucocorticoid Cell Priming Enhances Transfection Outcomes in Adult Human Mesenchymal Stem Cells

Abstract

Human mesenchymal stem cells (hMSCs) are one of the most widely researched stem cell types with broad applications from basic research to therapeutics, the majority of which require introduction of exogenous DNA. However, safety and scalability issues hinder viral delivery, while poor efficiency hinders nonviral gene delivery, particularly to hMSCs. Here, we present the use of a pharmacologic agent (glucocorticoid) to overcome barriers to hMSC DNA transfer to enhance transfection using three common nonviral vectors. Glucocorticoid priming significantly enhances transfection in hMSCs, demonstrated by a 3-fold increase in efficiency, 4-15-fold increase in transgene expression, and prolonged transgene expression when compared to transfection without glucocorticoids. These effects are dependent on glucocorticoid receptor binding and caused in part by maintenance of normal metabolic function and increased cellular (5-fold) and nuclear (6-10-fold) DNA uptake over hMSCs transfected without glucocorticoids. Results were consistent across five human donors and in cells up to passage five. Glucocorticoid cell priming is a simple and effective technique to significantly enhance nonviral transfection of hMSCs that should enhance their clinical use, accelerate new research, and decrease reliance on early passage cells.

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

“…Plasmids Bernasconi et al, 1997;Braun et al, 1999;Chen et al, 2011;Choi & Lee, 2005;Jain et al, 1999;Koster et al, 2002;Köster et al, 2006;Lin et al, 2003;Nair et al, 2002) compared to those seen here in hMSCs. Our results showing increased transgenic enzyme activity in addition to increased transgenic protein production (all relative to VCs) are probably not specific to only luciferase transgenes because we previously reported DEX-priming yielded 4-7-fold increases in transgenic β-galactosidase enzyme activity, whereas also increasing transgenic EGFP mean fluorescence intensity by about two-fold, all relative to VC (Kelly et al, 2016). Our results showing increased transgenic enzyme activity in addition to increased transgenic protein production (all relative to VCs) are probably not specific to only luciferase transgenes because we previously reported DEX-priming yielded 4-7-fold increases in transgenic β-galactosidase enzyme activity, whereas also increasing transgenic EGFP mean fluorescence intensity by about two-fold, all relative to VC (Kelly et al, 2016).…”

“…Although DEX increased transgenic luciferase activity normalized to total protein by about 10-fold in transfected hMSCs, DEX only increased the amount of transgenic luciferase protein by about two-fold, as quantified by western blot analysis normalized to total protein (Figure 1c,d), suggesting that DEX-priming somehow increases transgenic enzyme activity downstream of transgenic protein synthesis. It is likely that hMSC transfection could be primed with many other Gc drugs in addition to DEX, as our previous work demonstrated that priming with cortisol similarly resulted in significant enhancement of transgene expression in transfected hMSCs (Kelly et al, 2016). It is likely that hMSC transfection could be primed with many other Gc drugs in addition to DEX, as our previous work demonstrated that priming with cortisol similarly resulted in significant enhancement of transgene expression in transfected hMSCs (Kelly et al, 2016).…”

“…Most Gc effects within cells are mediated by binding and activation of the GR and we previously demonstrated that binding of the GR is required for DEX-mediated enhancement of transgene expression in hMSCs (Kelly et al, 2016). Since DEX has been shown to alter nuclear membrane permeability (Kastrup, Oberleithner, Ludwig, Schafer, & Shahin, 2006;Shahin, 2006;Shahin et al, 2005), and the GR has been shown to interact with similar transport and nuclear import pathways as pDNA, we quantified the number of plasmid copies in transfected hMSCs and isolated nuclei, with and without DEX-priming.…”

“…Specifically, we investigated anti-inflammatory glucocorticoid (Gc) drugs as priming candidates in hMSCs due to reports in other cell types that Gc can increase transfection efficiency by increasing plasmid DNA (pDNA) nuclear internalization or reducing the inflammatory response to transfection (Braun et al, 1999;Kim, Kim, Bae, Choi, & Lee, 2009). In hMSCs, derived from bone marrow stromal cells (BMSCs) of multiple donors, we showed that 90 to 360 nM dexamethasone (DEX), a Gc drug, delivered 0-30 min before delivery of pDNA lipoplexes increased the transgenic luciferase activity about 10-fold, increased transgenic enhanced green fluorescent protein (EGFP) mean fluorescence intensity of transfected cells about two-fold, increased transfection efficiency about three-fold (i.e., percent of EGFP + transfected cells), increased duration of transgene expression, and ameliorated transfection-induced metabolic decline, all while retaining differentiation capacity (Kelly, Plautz, Zempleni, & Pannier, 2016). Others have reported similar enhancement of transfection using steroids in other cell types.…”

“…Since DEX-priming of hMSC transfection is mediated by binding of the glucocorticoid receptor (GR; Kelly et al, 2016), which makes use of similar intracellular transport mechanisms as transfected pDNA does (Davies, Ning, & Sánchez, 2002;Dhanoya, Wang, Keshavarz-Moore, Fassati, & Chain, 2013;Echeverria et al, 2009;Galigniana, Harrell, O'Hagen, Ljungman, & Pratt, 2004;Harrell et al, 2004;Lachish-Zalait et al, 2009), and GR modulates gene expression related to antiinflammatory and stress pathways (Ratman et al, 2013), we analyze nonviral gene delivery barriers and pathways in hMSCs including internalization, cytoplasmic transport, nuclear translocation, transgene transcription, translation, and cellular stress response, along with the effects of DEX-priming on each of these barriers and pathways. We also explore the effects of different pDNA regulatory sequences on transgene expression and DEX-priming.…”