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. 2020 Jul 23:18:713-722.
doi: 10.1016/j.omtm.2020.07.014. eCollection 2020 Sep 11.

Glucocorticoid Priming of Nonviral Gene Delivery to hMSCs Increases Transfection by Reducing Induced Stresses

Andrew Hamann  1 , Tyler Kozisek  1 , Kelly Broad  1 , Angela K Pannier  1
Affiliations

Glucocorticoid Priming of Nonviral Gene Delivery to hMSCs Increases Transfection by Reducing Induced Stresses

Andrew Hamann et al. Mol Ther Methods Clin Dev. .

Abstract

Human mesenchymal stem cells (hMSCs) are under study for cell and gene therapeutics because of their immunomodulatory and regenerative properties. Safe and efficient gene delivery could increase hMSC clinical potential by enabling expression of transgenes for control over factor production, behavior, and differentiation. Viral delivery is efficient but suffers from safety issues, while nonviral methods are safe but highly inefficient, especially in hMSCs. We previously demonstrated that priming cells with glucocorticoids (Gcs) before delivery of DNA complexes significantly increases hMSC transfection, which correlates with a rescue of transfection-induced metabolic and protein synthesis decline, and apoptosis. In this work, we show that transgene expression enhancement is mediated by transcriptional activation of endogenous hMSC genes by the cytosolic glucocorticoid receptor (cGR) and that transfection enhancement can be potentiated with a GR transcription-activation synergist. We demonstrate that the Gc-activated cGR modulates endogenous hMSC gene expression to ameliorate transfection-induced endoplasmic reticulum (ER) and oxidative stresses, apoptosis, and inflammatory responses to prevent hMSC metabolic and protein synthesis decline, resulting in enhanced transgene expression after nonviral gene delivery to hMSCs. These results provide insights important for rational design of more efficient nonviral gene delivery and priming techniques that could be utilized for clinical hMSC applications.

Keywords: dexamethasone; glucocorticoid; lipoplex; mesenchymal stem cell; nonviral gene delivery; priming; transfection.

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Figures

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Graphical abstract
Figure 1
Figure 1
Priming hMSCs with the Gc Drug DEX Significantly Enhances Nonviral Gene Delivery (A and B) 150 nM DEX treatment 25 min prior to transfection with lipid reagent, Lipofectamine (LF) 3000, complexed with pDNA encoding a fusion protein of luciferase and enhanced green fluorescent protein (EGFP), increased (A) transgenic luciferase expression from 4- to 8-fold as measured in relative light units normalized per milligram of cellular protein (RLU/mg protein) and (B) increased transfection efficiency (i.e., the proportion of hMSCs expressing EGFP) by about 2-fold as quantified by fluorescent microscopy, all relative to vehicle control (VC) treatment, in D1 AMSCs, D3 AMSCs, D2 BMSCs, and D4 BMSCs. Data are plotted as mean ± SEM (n = 3). Asterisks denote significance to VC conditions: ∗p ≤ 0.05; ∗∗p ≤ 0.01. Fold change increase over VC conditions.
Figure 2
Figure 2
Cell-Impermeable Glucocorticoid Does Not Enhance hMSC Transfection Cell-permeable free cortisol (Cort), capable of binding cytosolic glucocorticoid receptor (cGR), delivered with bovine serum albumin (BSA) significantly increases transgenic luciferase expression in transfected hMSCs over the VC, but membrane-impermeable BSA-Cort conjugate, only capable of binding membranous (mGR), does not increase luciferase expression. hMSCs were primed with compounds 25 min prior to transfection with lipid-pDNA complexes and lysed for analysis after 48 h. (A–D) Free Cort treatment resulted in 3- to 14-fold increases in transgene expression, normalized to total cellular protein, over VC in (A) D1 BMSCs, (B) D2 BMSCs, (C) D3 BMSCs, and (D) D1 AMSCs. Data are plotted as mean ± SEM (n = 3) of luciferase luminescence relative light units per milligram of total protein (RLU/mg protein). Asterisks denote significance to VC conditions: ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001. Fold change increase over VC conditions.
Figure 3
Figure 3
hMSC Transfection Is Not Enhanced by Glucocorticoid without Transcription-Activation Properties DEX, which induces GR to both induce and repress endogenous gene transcription, significantly increases transgenic luciferase expression in transfected hMSCs over the VC, but CpdA, which only induces GR to repress endogenous gene transcription, does not increase luciferase expression over VC. hMSCs were primed with compounds 25 min prior to transfection with LF 3000 pDNA complexes and lysed for analysis after 48 h. (A–D) D1 BMSCs (A); D3 BMSCs (B); D1 AMSCs (C); and D2 AMSCs (D). Data are plotted as mean ± SEM (n = 3) of luciferase luminescence relative light units per milligram of total protein (RLU/mg protein). Asterisks denote significance to VC conditions: ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001. Fold change increase over VC conditions.
Figure 4
Figure 4
DEX Transfection Priming Is Potentiated by Compound that Inhibits GR Nuclear Export (A–D) 150 nM DEX increases transgenic luciferase expression in transfected hMSCs over VC; and 150 nM DEX with varying doses of KPT-330 (KPT), a compound that inhibits nuclear export of the GR, significantly increases transgene expression from 2- to 4-fold over DEX alone in (A) D2 BMSCs, (B) D3 BMSCs, (C) D1 AMSCs, and (D) D2 AMSCs. hMSCs were primed with compounds 25 min prior to transfection with LF 3000 pDNA complexes and lysed for analysis after 48 h. Data are plotted as mean ± SEM (n = 3) of luciferase luminescence relative light units per mg of total protein (RLU/mg protein). Asterisks denote significance to 150 nM DEX-alone conditions: ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001. Fold change increase over 150 nM DEX-alone conditions.
Figure 5
Figure 5
DEX Priming Decreases hMSC Oxidative Stress Induced by Transfection (A–D) hMSCs were either untreated, transfected with 150 nM DEX priming, or transfected with VC priming. After 48 h, cellular reactive oxygen species (ROS) and nuclei were stained to calculate the fraction of hMSCs experiencing oxidative stress using fluorescent microscopy in (A) D1 AMSCs, (B) D2 AMSCs, (C) D3 AMSCs, and (D) D4 BMSCs. Data are plotted as mean ± SEM (n = 3). Asterisks denote significance to designated conditions: ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001.
Figure 6
Figure 6
Transfection and DEX Priming Modulates Endogenous hMSC Genes (A–D) Transfection and DEX priming modulates expression of genes related to endoplasmic reticulum (ER) stress, apoptosis, oxidative stress, and inflammation in hMSCs, as quantified by qRT-PCR 24 h after transfection. In D2 BMSCs, D3 BMSCs, D1 AMSCs, and D2 AMSCs: (A) ER stress-induced and apoptosis mediator CCAAT-enhancer-binding protein homologous protein (CHOP) messenger RNA (mRNA) was upregulated by transfection when treated with only VC and downregulated by DEX treatment when transfected relative to VC; (B) antioxidant protein metallothionein mRNA was upregulated by transfection when treated with VC and further upregulated by transfection with DEX; (C) inflammatory cytokine interleukin-6 (IL-6) mRNA was upregulated by transfection when treated with VC and downregulated by DEX treatment when transfected relative to VC; and (D) inflammatory enzyme cyclooxygenase-2 (COX2) mRNA was upregulated by transfection when treated with VC and downregulated by DEX treatment when transfected relative to VC. Data are plotted as mean ± SEM (n = 3). Pound symbols denote significance to untransfected conditions: #p ≤ 0.05; ##p ≤ 0.01; ###p ≤ 0.001; ####p ≤ 0.0001. Asterisks denote significance to VC conditions: ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001.
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References

    1. Chamberlain G., Fox J., Ashton B., Middleton J. Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing. Stem Cells. 2007;25:2739–2749. - PubMed
    1. Maxson S., Lopez E.A., Yoo D., Danilkovitch-Miagkova A., Leroux M.A. Concise review: role of mesenchymal stem cells in wound repair. Stem Cells Transl. Med. 2012;1:142–149. - PMC - PubMed
    1. Singer N.G., Caplan A.I. Mesenchymal stem cells: mechanisms of inflammation. Annu. Rev. Pathol. 2011;6:457–478. - PubMed
    1. Ren G., Zhang L., Zhao X., Xu G., Zhang Y., Roberts A.I., Zhao R.C., Shi Y. Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide. Cell Stem Cell. 2008;2:141–150. - PubMed
    1. Baksh D., Song L., Tuan R.S. Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy. J. Cell. Mol. Med. 2004;8:301–316. - PMC - PubMed

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