ABSTRACT
BACKGROUND: Wharton's Jelly-derived mesenchymal stem cells are relatively primitive stem cells that are ideal vectors for gene therapy. However, there is a lack of studies on the conditions for the electrotransfection of Wharton's Jelly-derived mesenchymal stem cells. Therefore, exploring the optimal conditions for the electrotransfection of Wharton's Jelly-derived mesenchymal stem cells occupies an important position. OBJECTIVE: To investigate the effects of different electroporation conditions on the transfection efficiency of Wharton's Jelly-derived mesenchymal stem cells, and to explore the optimal conditions for cell electroporation. METHODS: By controlling the transfection conditions such as voltage, pulse duration and cell status, EEV-EGFP plasmids were transfected into Wharton's Jelly-derived mesenchymal stem cells by electroporation under different conditions. Transfection efficiency was detected by flow cytometry. RESULTS AND CONCLUSION: (1) The transfection efficiency was intended to increase when the voltage ranged from 125 V to 150 V, and the maximum transfection efficiency was obtained when the voltage was 150 V. However, when the voltage was further increased to 170 V, the transfection efficiency began to decrease considerably. (2) The maximum transfection efficiency was obtained when the pulse duration was 5.0 ms, while it was certainly decreased when the pulse duration was 2.5 and 7.5 ms. (3) The transfection efficiency of the cells cultured under normoxia was higher than that under hypoxic culture. These findings reveal that normally cultured Wharton's Jelly-derived mesenchymal stem cells can achieve higher electroporation efficiency via two pulse sessions at a voltage of 150 V, pulse duration of 5.0 ms, and pulse interval time of 50 ms.
ABSTRACT
This study was aimed to investigate the transfection efficacy of recombinant adeno-associated virus 2/1 (rAAV2/1) on bone marrow mesenchymal stem cells (BMMSCs) at different multiplicities of infection (MOI) and time, and effect of transfection on growth of rat BMMSCs. The rat BMMSCs cultured in vitro were transfected by using rAAV2/1 with enhanced green fluorescent protein (rAAV2/1-EGFP) at MOI of 1 x 10(4), 1 x 10(5) and 1 x 10(6); the EGFP expression was observed by fluorescent microscopy at 3, 7 and 14 days. The viability, proliferation multiple, differentiation ability of daughter cells were detected for evaluating the effect of rAAV2/1 on survival, proliferation and differentiation of BMMSCs and the fluorescence index (FI) were determined by flow cytometry. The results indicated that after transfection with rAAV2/1 for 24 hours the green fluorescence in BMMSCs were observed, but also the fluorescence gradually was enhanced along with prolonging of time, and reached to steady level after 7 days; the viability, proliferation multiple, differentiation ability of BMMSCs transfected by rAAV2/1-EGFP at different MOI showed no significant changes at 3,7 and 14 days (p > 0.05), meanwhile at same MOI the proliferation multiple obviously increased in comparison between 7 day vs 3 day and 14 days vs 7 days (p < 0.01). The flow cytometric detection showed that the transfection efficacy of rAAV2/1-EGFP on BMMSCs and FI increased significantly as the multiplicity of infection and culture time increased (p < 0.05). It is concluded that rAAV2/1-EGFP is able to transfect into BMMSCs effectively, but the transfection efficiency and fluorescence index increase significantly along with increase of multiplicity of infection and culture time. rAAV2/1-EGFP do not affect viability, proliferation multiple and differentiation ability of BMMSCs. rAAV2/1 is a kind of active vector for gene transfer to reform BMMSCs.