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/cm(2) . 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.

Fig 1

Characterization of PEI/DNA complexes. Plasmid DNA was increasingly retarded by PEI with the increase of N/P ratio, until the complete retardation at N/P ratio 1 (A). PEI/DNA complex size was reduced, whereas ζ potential was increased with the increase of N/P ratio from 0.5 to 12 (B).

Fig 2

Representative result of the phenotypic characterization of hMSCs (A). The cells showed positive expression of MSC markers CD29, CD44, CD73, CD105 and negative expression of haematopoietic stem cell markers CD45. The hMSCs had a spindle-shaped morphology (B) (Bar = 100 μM).

Fig 3

The cell viability decreased with the increase of N/P ratio and/or DNA dosage. The result was given as average ± S.D. (n = 8).

Fig 4

Representative transfection efficiencies of hMSCs (donor age 67, male) with various N/P ratio and DNA dosage (A), and the histograms of FACS analysis at N/P ratio 2 and various DNA dosage (B). (C–E): Representative GFP expression of transfected hMSCs under optimal transfection conditions. Fluorescence image (C), phase-contrast image (D) and merged (E) (Bar = 100 μM).

Fig 5

The effect of cell resource on transfection efficiency. No correlation was observed between transfection efficiency and donors’ age according to Spearman’s rank correlation analysis (A). Independent-samples t-test revealed there was no significant difference of transfection efficiency between middle-age (n = 9) and old-age (n = 21) groups (B), and between female (n = 7) and male (n = 23) groups (C). The result was expressed as average ± S.E.M. (standard error of the mean).

Fig 6

Association between transfection efficiency and the cell percentage in S phase. Two subpopulations were found and linear regression indicated the trend towards the correlation between transfection efficiency and the cell percentage in S phase in each subpopulation (A). A representative histogram of cell cycle analysis via PI staining and FACS detection (B). Phenotypic characterization showed no significant difference between these two subpopulations (C) [Average ± S.E.M, n = 4 (P1) and n = 3 (P2)].

Fig 7

VEGF165 expression level of untransfected cells (1.84 ± 0.11 pg/μg total protein), naked plasmid DNA transfected cells (1.94 ± 0.11 pg/μg total protein) and PEI/DNA transfected cells (3.49 ± 0.52 pg/μg total protein). The result was given as average ± S.E.M. (standard error of the mean). (n = 8, *P < 0.05 by paired-samples t-test).