Extended and stable gene expression via nucleofection of MIDGE construct into adult human marrow mesenchymal stromal cells

Abstract

Human mesenchymal stromal cell (hMSC) is a potential target for cell and gene therapy-based approaches against a variety of different diseases. Whilst cationic lipofection has been widely experimented, the Nucleofector technology is a relatively new non-viral transfection method designed for primary cells and hard-to-transfect cell lines. Herein, we compared the efficiency and viability of nucleofection with cationic lipofection, and used the more efficient transfection method, nucleofection, to deliver a construct of minimalistic, immunologically defined gene expression encoding the erythropoietin (MIDGE-EPO) into hMSC. MIDGE construct is relatively safer than the viral and plasmid expression systems as the detrimental eukaryotic and prokaryotic gene and sequences have been eliminated. Using a plasmid encoding the luciferase gene, we demonstrated a high transfection efficiency using the U-23 (21.79 ± 1.09%) and C-17 (5.62 ± 1.09%) pulsing program in nucleofection. The cell viabilities were (44.93 ± 10.10)% and (21.93 ± 5.72)%, respectively 24 h post-nucleofection. On the other hand, lipofection treatment only yielded less than 0.6% efficiencies despite showing higher viabilities. Nucleofection did not affect hMSC renewability, immunophenotype and differentiation potentials. Subsequently, we nucleofected MIDGE-EPO using the U-23 pulsing program into hMSC. The results showed that, despite a low nucleofection efficiency with this construct, the EPO protein was stably expressed in the nucleofected cells up to 55 days when determined by ELISA or immunocytochemical staining. In conclusion, nucleofection is an efficient non-viral transfection approach for hMSC, which when used in conjunction with a MIDGE construct, could result in extended and stable transgene expression in hMSC.

Human Mesenchymal Stem Cells Expressing Erythropoietin Enhance Survivability of Retinal Neurons Against Oxidative Stress: An In Vitro Study

“…In addition to the widely distributed occurrence of EPO and its associated EPO receptors (EPOR) in retinal tissue (Shirley Ding et al, 2016 ), as well as the ability of EPO to cross the BRB, studies found that EPO reacts with the damaged photoreceptors by crosslinking with the EPOR site (Grimm et al, 2002 ). Furthermore, accumulating findings have also outlined the promising use of EPO (Zhu et al, 2009 ; Mok et al, 2012 ; Boesch et al, 2014 ) in the treatment of retinal degenerative diseases, such as AMD (Wang et al, 2009 ), retinitis pigmentosa, and diabetic retinopathy (McVicar et al, 2011 ). Coupling EPO with MSC therapy also increases the probability of successful MSC transplantation in a harsh microenvironment, which will lead to amplifying the regenerative potential of MSCs.…”

Genetic engineering of mesenchymal stem cells by non-viral gene delivery

“…For MSC transfection, nucleofection has been proved to present better transfection results than conventional electroporation [119] and transfection mediated by cationic lipids [3,59,113]. The nucleofection efficiency of human bone marrow derived MSCs could reach up to around 70% ( Figure 2) [6].…”

Empowering Mesenchymal Stem Cells for Ocular Degenerative Disorders

“…A substantial advance in our understanding of the regulatory machinery and beneficial secretory proteins of MSCs have paved the way for further development of the technique. Harnessing the potential of biomaterials and tissue engineering [12,25,26,27,28,29], nanotechnology [30,31,32,33], and genome engineering [10,34,35,36,37,38,39,40,41,42,43] to maximize MSCs therapeutic insight for stem cell replacement therapy holds potential for further leaps in using MSC in stem cell therapy. For a clinical translatable stem cell therapy for ocular degenerative disorders, integration of tissue engineering approaches will overcome limitations associated with low transplanted cell survivability [21,22] and cell dispersion [23], and further encourage a targeted delivery system in the transplanted MSCs.…”