Polyethylenimine-mediated gene delivery into human bone marrow mesenchymal stem cells from patients.
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.
Wang
Weiwei
W
Department of Cardiac Surgery, University of Rostock, Rostock, Germany.
Li
Wenzhong
W
Ou
Lailiang
L
Flick
Eva
E
Mark
Peter
P
Nesselmann
Catharina
C
Lux
Cornelia A
CA
Gatzen
Hans-Heinrich
HH
Kaminski
Alexander
A
Liebold
Andreas
A
Lützow
Karola
K
Lendlein
Andreas
A
Li
Ren-Ke
RK
Steinhoff
Gustav
G
Ma
Nan
N
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004247:DNA / Q000378:metabolism; D005260:Female; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008875:Middle Aged; D010641:Phenotype; D011094:Polyethyleneimine / Q000494:pharmacology*; D016196:S Phase / Q000187:drug effects; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D049452:Green Fluorescent Proteins; D011094:Polyethyleneimine; D004247:DNA