Highly Efficient Transfection of Human Marrow Stromal Cells by Nucleofection

doi:10.1016/j.transproceed.2005.03.047

Transplantation Proceedings

Volume 37, Issue 5, June 2005, Pages 2290-2292

https://doi.org/10.1016/j.transproceed.2005.03.047 Get rights and content

Abstract

Human marrow stromal cells (hMSCs) are an attractive source for autologous cell and gene therapies. In this study, we developed a highly efficient transfection method for hMSCs. Although they tend to show efficient gene delivery, nonviral vectors offer several advantages over viral vectors for gene therapies. They are inexpensive to produce and suitable to adopt particularly with respect to little or no specific immune responses; they are simple to use; they entail easier large-scale production; and they have a high degree quality control. hMSCs and rat marrow stromal cells were transfected with the plasmid pEGFP-N1 that encoded a green fluorescent protein component by using two nonviral methods: nucleofection and electroporation. Nucleofection provided a much better rate of transfer than electroporation particularly in hMSCs.

Section snippets

Plasmid DNA

pEGFP-N1 obtained from Clontech (Palo Alto, Calif, USA) was used to gauge transfection efficiency. pEGFP-N1 propagated in Escherichia coli strain DH5α (Toyobo, Osaka, Japapn) was purified with the Qiagen EndoFree plasmid kit following the manufacturer’s instructions (Qiagen, Hilden, Germany).

Cell Culture

hMSCs were isolated from 10-mL aspirates of bone marrow from the iliac crest. After centrifugation, the cells at the interface were distributed in αMEM. Passages were performed when cells approached

Electroporation

At 24 hours after electroporation, 13.0% of hMSCs and 6.8% of rMSCs expressed GFP (Fig 1). The highest efficiency of the transfection was obtained at 600 V and 100 μs in both hMSCs and rMSCs without any visible toxic side effects.

Nucleofection

At 24 hours after nucleofection, 41.8% of hMSCs and 9.9% of rMSCs expressed GFP (Fig 2). The highest efficiency of the transfection was obtained at program A-23 without significant cell toxicity.

Discussion

In general, nonviral DNA transfection methods are inefficient, particularly in nondividing cells, since there is only limited DNA trafficking to the nucleus where transcription occurs.2, 3 In our initial experiments, we also tested the transfection efficiency of hMSCs and rMSCS with cationic lipids (LipfectAMINE PLUS, invitrogen) transfection regents, using the pEGFP-N1 vector we observed low transfection efficiency (data not shown).

However, a new technology—nucleofection—has been developed

References (5)

P. Lenz et al.
Nucleoporation of dendritic cellsefficient gene transfer by electroporation into human monocyte-derived dendric cells
FEBS Lett
(2003)
G. Nikcevic et al.
Improved transfection efficiency of cultured human cells
Cell Biol Int
(2003)

There are more references available in the full text version of this article.

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Hematopoietic and stem cellHighly Efficient Transfection of Human Marrow Stromal Cells by Nucleofection