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. 2019 Feb;116(2):427-443.
doi: 10.1002/bit.26870. Epub 2018 Dec 7.

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

Andrew Hamann  1 , Kelly Broad  1 , Albert Nguyen  1 , Angela K Pannier  1
Affiliations

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

Andrew Hamann et al. Biotechnol Bioeng. 2019 Feb.

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.

Keywords: dexamethasone; glucocorticoid; human mesenchymal stem cells; nonviral gene delivery; priming; transfection.

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Conflict of interest statement

The authors declare that there are no conflicts of interest.

Figures

Figure 1
Figure 1
Dexamethasone (DEX)‐priming significantly increases hMSC transgenic luciferase amount and further increases transgenic luciferase activity. hMSCs were primed with 150 nM DEX 25 min before transfection with Lipofectamine‐3000 plasmid DNA complexes, and lysed for analysis after 48 hr. DEX‐treated hMSCs displayed about 10‐fold increases in the luciferase activity, normalized to total cellular protein, over ethanol (EtOH) treated cells in (a) Donor 4 (D4) BMSCs and (b) D1 AMSCs. In contrast, DEX only increased transgenic luciferase amount by about two‐fold relative to EtOH, as quantified by western blot analysis, in transfected (c) D5 BMSCs, and (d) D1 AMSCs. Luciferase activity data plotted as mean ± SEM (n = 3) of luciferase luminescence relative light units per mg of total protein (RLU/mg protein). Relative luciferase amount data from western blot is normalized to total protein and plotted as mean ± SEM (n = 2). Asterisks (*) denote significance to EtOH conditions (*p ≤ 0.05; ***p ≤ 0.001). Fold change increase over EtOH conditions shown in parentheses. AMSCs: adipose tissue derived from mesenchymal stem cells; BMSCs: bone marrow derived from mesenchymal stem cells; hMSCs: human mesenchymal stem cells; SEM: standard error of mean
Figure 2
Figure 2
Dexamethasone (DEX)‐priming does not increase plasmid DNA cellular or nuclear internalization in hMSCs. hMSCs were primed with 150 nM DEX or ethanol (EtOH) 25 min before transfection with Lipofectomine‐3000 plasmid DNA complexes. After 48 hr, whole cells and isolated nuclei were lysed and the number of internalized plasmids per cell and per nuclei was determined by qPCR. DEX treatment did not increase the number of plasmids internalized by whole cells or nuclei in (a) Donor 3 (D3) BMSCs, (b) D4 BMSCs, (c) D1 AMSCs, (d) or D2 AMSCs. Data is plotted as means ± SEM (n = 3). Asterisks denote significance to EtOH conditions (*p ≤ 0.05). AMSCs: adipose tissue derived from mesenchymal stem cells; BMSCs: bone marrow derived from mesenchymal stem cells; hMSCs: human mesenchymal stem cells; qPCR: quantitative polymerase chain reaction; SEM: standard error of mean
Figure 3
Figure 3
Cytoplasmic transport inhibition moderately decreases the hMSC transgenic luciferase activity enhancement induced by dexamethasone (DEX)‐priming. hMSCs were treated with 1 μM nocodazole (Noco) or 12 μM ciliobrevin (Cilio), specific inhibitors of microtubule polymerization or dynein motor protein motion, respectively, 1 hr before priming with 150 nM DEX 25 min before transfection with Lipofectamine plasmid DNA complexes. Noco and Cilio in the absence of DEX (+/−/− and −/+/−) had little effect on luciferase transgene activity compared to control cells treated with no inhibitor or DEX (−/−/−), but decreased fold‐change enhancement by DEX‐priming (+/−/+ and −/+/+) relative to control cells treated with DEX, but no inhibitor (−/−/−) in (a) Donor 1 (D1) BMSCs, (b) D4 BMSCs, (c) D2 AMSCs, and (d) D3 AMSCs. Data is plotted as mean of fold changes relative to control cells not treated with inhibitors or DEX (−/−/−) ± SEM (n = 3). Asterisks (*) denote significance to −/−/− cells and pounds (#) denote significance to control cells treated with DEX, but no inhibitor (−/−/+; ## p ≤ 0.01; ### p ≤ 0.001). One‐way ANOVA was performed, as comparisons were made between a control condition and conditions in which one independent variable was manipulated (i.e. inhibitor vs. no inhibitor, whereas not varying whether or not DEX was present). Appropriate controls were treated with ethanol in lieu of inhibitor or DEX. AMSC: adipose tissue derived from mesenchymal stem cells; BMSC: bone marrow derived from mesenchymal stem cells; hMSCs: human mesenchymal stem cells; SEM: standard error of mean
Figure 4
Figure 4
Nuclear import inhibition does not reduce transgenic luciferase activity enhancement induced by dexamethasone (DEX)‐priming. hMSCs were treated with either 1 μM ivermectin (IV) or 25 μM importazole (IM), specific inhibitors of nuclear import mediated by either the importin α/β pathway or the Importin β pathway, respectively, 1 hr before priming with 150 nM DEX 25 min before transfection with Lipofectamine plasmid DNA complexes. IV in the absence of DEX (+/−/−) showed moderate decreases in transgenic luciferase activity, wh IM in the absence of DEX (−/+/−) dramatically decreased transgenic luciferase activity relative to control cells not treated with inhibitor or DEX (−/−/−), but neither inhibitor decreased fold‐change enhancement induced by DEX (+/−/+ and −/+/+) relative to control cells treated with DEX, but no inhibitor (−/−/+) in (a) Donor 3 (D3) BMSCs, (b) D4 BMSCs, (c) D1 AMSCs, and (d) D2 AMSCs. Data are plotted as mean of fold changes relative to control cells not treated with inhibitors or DEX (−/−/−) ± SEM (n = 3). Asterisks denote significance to −/−/− cells (*p ≤ 0.05; *** p ≤ 0.001) and pounds (#) denote significance to control cells treated with DEX, but no inhibitor (−/−/+; ### p ≤ 0.001). One‐way analysis of variance was performed, as comparisons were made between a control condition and conditions in which one independent variable was manipulated (i.e., inhibitor vs. no inhibitor, whereas not varying whether or not DEX was present). Appropriate controls were treated with ethanol in lieu of inhibitor or DEX. AMSCs: adipose tissue derived from mesenchymal stem cells; BMSCs: bone marrow derived from mesenchymal stem cells; SEM: standard error of mean
Figure 5
Figure 5
Plasmid sequence modifications do not affect hMSC transgenic luciferase activity enhancement by dexamethasone (DEX)‐priming. hMSCs were transfected with Lipofectomine‐3000 complexed with modifications of pEGFP‐Luc expressing luciferase transgene driven by cytomegalovirus promoter with SV40 enhancer (CMV [unmodified pEGFP‐Luc]) or without SV40 enhancer (CMV‐NoSV), with consensus glucocorticoid response element added upstream of CMV promoter (CMV‐GRE), or with CMV promoter replaced by elongation factor 1 α promoter (EF1α) or rous sarcoma virus promoter (RSV). Cells were treated with 150 nM DEX or ethanol (EtOH) 25 min before transfection. Sequence modifications had variable effect on transgenic luciferase activity levels, but did not prevent enhancement by DEX‐priming in (a) Donor 2 (D2) BMSCs, (b) D4 BMSCs, or (c) D2 AMSCs. Data is plotted as mean of fold changes relative to cells transfected with CMV plasmid and not primed with DEX ± SEM (n = 3). AMSC: adipose tissue derived from mesenchymal stem cell; BMSC: bone marrow derived from mesenchymal stem cell; CMV: cytomegalovirus; hMSCs: human mesenchymal stem cells; SEM: standard error of mean
Figure 6
Figure 6
Dexamethasone (DEX)‐priming does not increase transgene (i.e., EGFP) messenger RNA (mRNA) levels. hMSCs were primed with 150 nM DEX or ethanol (EtOH) 25 min before transfection with plasmid DNA Lipofectomine‐3000 complexes. After 48 hr, total RNA was extracted and relative copies of transgene mRNA was determined by qRT‐PCR. DEX‐priming did not significantly affect relative transgene mRNA transcript amount in (a) Donor 4 (D4) BMSCs, (b) D5 BMSCs, (c) D2 AMSCs, or (d) D3 AMSCs. Data is plotted as means ± SEM (n = 3). Statistical significance was considered at p < 0.05. AMSC: adipose tissue derived from mesenchymal stem cell; BMSC: bone marrow derived from mesenchymal stem cell; hMSC: human mesenchymal stem cell; qRT‐PCR: quantitative real‐time polymerase chain reaction; SEM: standard error of mean
Figure 7
Figure 7
DEX‐priming ameliorates the reduction in protein synthesis induced by transfection in hMSCs. hMSCs were either untreated and untransfected (Ngtv), transfected with 150 nM DEX‐priming, or transfected with ethanol (EtOH) control priming. After 24 hr, cells were fed media containing O‐propargyl‐puromycin, which is incorporated into translated proteins over the next 24 hr and subsequently fluorescently labeled. Translation was measured in relative fluorescence units normalized to total cellular DNA fluorescently stained with Hoechst in (a) Donor 3 (D3) BMSCs, (b) D5 BMSCs, (c) D1 AMSCs, and (d) D3 AMSCs. Data is plotted as means ± SEM (n = 3). Asterisks denote significance compared to EtOH conditions (**p ≤ 0.01; ***p ≤ 0.001). AMSC: adipose tissue derived from mesenchymal stem cell; BMSC: bone marrow derived from mesenchymal stem cell; DEX: dexamethasone; hMSC: human mesenchymal stem cells; RFU: relative fluorescence unit; SEM: standard error of mean
Figure 8
Figure 8
DEX‐priming decreases apoptosis induced by transfection in hMSCs. hMSCs were either untreated and untransfected (Ngtv), transfected with 150 nM DEX‐priming, or transfected with ethanol (EtOH) control priming. After 48 hr, apoptotic cells stained with fluorescently labeled annexin were measured in relative fluorescent units normalized to total cellular DNA fluorescently stained with Hoechst to calculate plotted relative apoptosis in (a) D1 BMSCs, (b) D3 BMSCs, (c) D1 AMSCs, and (d) D3 AMSCs. Data is plotted as means ± SEM (n = 3). Asterisks denote significance compared to EtOH conditions (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001). AMSC: adipose tissue derived from mesenchymal stem cell; BMSC: bone marrow derived from mesenchymal stem cell; DEX: dexamethasone; hMSCs: human mesenchymal stem cells; SEM: standard error of mean
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