Polyethylenimine-mediated gene delivery into human bone marrow mesenchymal stem cells from patients

Abstract

Transplantation of mesenchymal stem cells (MSCs) derived from adult bone marrow has been proposed as a potential therapeutic approach for post-infarction left ventricular (LV) dysfunction. However, age-related functional decline of stem cells has restricted their clinical benefits after transplantation into the infarcted myocardium. The limitations imposed on patient cells could be addressed by genetic modification of stem cells. This study was designed to improve our understanding of genetic modification of human bone marrow derived mesenchymal stem cells (hMSCs) by polyethylenimine (PEI, branched with Mw 25 kD), one of non-viral vectors that show promise in stem cell genetic modification, in the context of cardiac regeneration for patients. We optimized the PEI-mediated reporter gene transfection into hMSCs, evaluated whether transfection efficiency is associated with gender or age of the cell donors, analysed the influence of cell cycle on transfection and investigated the transfer of therapeutic vascular endothelial growth factor gene (VEGF). hMSCs were isolated from patients with cardiovascular disease aged from 41 to 85 years. Optimization of gene delivery to hMSCs was carried out based on the particle size of the PEI/DNA complexes, N/P ratio of complexes, DNA dosage and cell viability. The highest efficiency with the cell viability near 60% was achieved at N/P ratio 2 and 6.0 μg DNA/cm2. The average transfection efficiency for all tested samples, middle-age group (<65 years), old-age group (>65 years), female group and male group was 4.32%, 3.85%, 4.52%, 4.14% and 4.38%, respectively. The transfection efficiency did not show any correlation either with the age or the gender of the donors. Statistically, there were two subpopulations in the donors; and transfection efficiency in each subpopulation was linearly related to the cell percentage in S phase. No significant phenotypic differences were observed between these two subpopulations. Furthermore, PEI-mediated therapeutic gene VEGF transfer could significantly enhance the expression level.

Fetal vs adult mesenchymal stem cells achieve greater gene expression, but less osteoinduction

“…In the evaluation of chemical-mediated transfection, we found that PEI provided a significant level of transfection efficiency in fMSCs. Our results are in accordance with a study by Wang et al [17] , which showed that genetic modification of human MSCs by PEI with a relatively low efficiency of 4% achieved biologically significant effects in vivo, with transfection efficiency proportional to cells in S phase [17] . Our analysis further supported the use of PEI as a transfection reagent in MSCs over other reagents.…”

The development of non-viral gene-activated matrices for bone regeneration using polyethyleneimine (PEI) and collagen-based scaffolds

“…Several groups (13,15,21) have previously demonstrated the high transfection efficiency of PEI in various cell types and cell lines. Although PEI has been reported as an efficient transfection tool in human adipose derived stem cells (25), human bone marrow MSCs [50] and a bone marrow derived stem cell line (27), collectively very little work has been reported using PEI as an efficient MSC transfection agent, despite its widespread availability as a vector. The majority of research conducted using gene delivery technology is carried out using readily available cells lines.…”

Adenovirus-mediated expression of the HO-1 protein within MSCs decreased cytotoxicity and inhibited apoptosis induced by oxidative stresses

“…Despite their several advantages, the low survival rate of MSCs following cell therapy is a major challenge that limits their efficacy in clinical settings (Wang et al 2010). Oxidative stresses and pro-apoptotic factors are well-known causes of MSCs death (Zhu et al 2006).…”