Robust, efficient, and practical electrogene transfer method for human mesenchymal stem cells using square electric pulses

Abstract

Mesenchymal stem cells (MSCs) are multipotent nonhematopoietic cells with the ability to differentiate into various specific cell types, thus holding great promise for regenerative medicine. Early clinical trials have proven that MSC-based therapy is safe, with possible efficacy in various diseased states. Moreover, genetic modification of MSCs to improve their function can be safely achieved using electrogene transfer. We previously achieved transfection efficiencies of up to 32% with preserved viability in rat MSCs. In this study, we further improved the transfection efficiency and transgene expression in human MSCs (hMSCs), while preserving the cells viability and ability to differentiate into osteoblasts and adipocytes by increasing the plasmid concentration and altering the osmotic pressure of the electrotransfer buffer. Using a square-wave electric pulse generator, we achieved a transfection efficiency of more than 80%, with around 70% viability and a detectable transgene expression of up to 30 days. Moreover, we demonstrated that this transfection efficiency can be reproduced reliably on two different sources of hMSCs: the bone marrow and adipose tissue. We also showed that there was no significant donor variability in terms of their transfection efficiency and viability. The cell confluency before electrotransfer had no significant effect on the transfection efficiency and viability. Cryopreservation of transfected cells maintained their transgene expression and viability upon thawing. In summary, we are reporting a robust, safe, and efficient protocol of electrotransfer for hMSCs with several practical suggestions for an optimal use of genetically engineered hMSCs for clinical application.

FIG. 1.

Reproducibility of the electrotransfer protocol. (a and b) Reproducible transfection efficiency of MSCs irrespective of donors or tissue source. BM-MSCs (a) and AT-MSCs (b) derived from eight different donors (n=5, p=1.00 (a) and p=0.33 (b), one-way ANOVA) were electrotransfected (1500 V/cm, 8 μg of plasmid in S-MEM). (c) Reproducible transfection efficiency of MSCs irrespective of the time post-thaw (n=3 except for group 168 hr where n=8, p=0.67, one-way ANOVA). (d) Decreased percentage of transfected cells when the pulse amplitude is increased beyond 1500 V/cm (n=3, p<0.05, one-way ANOVA). (e) Reproducible transfection efficiency of MSCs irrespective of pretreatment confluency (40% or 80%) (p=0.53, unpaired t-test). ( f–h) Reproducibility of the improved electrotransfer protocol (1500 V/cm, 52 μg of plasmid in S-MEM with 40% water). No effect of donor variability on transfection efficiency of BM-MSCs (f ) (n=3 except for BM-MSC#1 where n=4, p=0.75, one-way ANOVA) and AT-MSCs (h) (n=3, p=0.05, Kruskal–Wallis one-way ANOVA) derived from 10 different donors. (g) Slightly higher overall transfection efficiency in BM-MSCs as compared with AT-MSCs (79% vs. 69%, p<0.01, Mann–Whitney test). ANOVA, analysis of variance; AT-MSCs, adipose tissue-derived MSCs; BM-MSCs, bone marrow-derived MSCs; MSCs, mesenchymal stem cells.

FIG. 2.

Increase in transfection efficiency using increasing concentration of plasmid (n=3 except for group 8 μg, where n=16, p<0.0001, one-way ANOVA with Bonferroni's multiple comparison test).

FIG. 3.

(a) Increase of median GFP expression per cell by using S-MEM with 40% of water. (b) Increase in percentage of cells expressing GFP after addition of 40% water (n=3 except for group DNA+H₂O+pulses, where n=7, p<0.01 (a) and p<0.0001 (b), one-way ANOVA with Bonferroni's multiple comparison test).

FIG. 4.

Kinetics of GFP expression in the MSCs electrotransfected under optimized conditions. MSCs were pulsed in S-MEM with 40% water and 52 μg of plasmid (white squares) or in S-MEM with 8 μg of plasmid (black diamonds). Optimization of the electrotransfer protocol extends both the level and the duration of GFP expression. The percentage of transfected cells remained stable for a week (a). However, the intensity of GFP expression started to decrease at a much earlier time point (b).

FIG. 5.

Morphology and GFP expression of transfected cells at day six after thawing (71 days post-EGT). (a) Fluorescence image (GFP). (b) Phase-contrast image.