A Comparative Study of Non-Viral Gene Delivery Techniques to Human Adipose-Derived Mesenchymal Stem Cell

Abstract

Mesenchymal stem cells (MSCs) hold tremendous potential for therapeutic use in stem cell-based gene therapy. Ex vivo genetic modification of MSCs with beneficial genes of interest is a prerequisite for successful use of stem cell-based therapeutic applications. However, genetic manipulation of MSCs is challenging because they are resistant to commonly used methods to introduce exogenous DNA or RNA. Herein we compared the effectiveness of several techniques (classic calcium phosphate precipitation, cationic polymer, and standard electroporation) with that of microporation technology to introduce the plasmid encoding for angiopoietin-1 (ANGPT-1) and enhanced green fluorescent protein (eGFP) into human adipose-derived MSCs (hAD-MSCs). The microporation technique had a higher transfection efficiency, with up to 50% of the viable hAD-MSCs being transfected, compared to the other transfection techniques, for which less than 1% of cells were positive for eGFP expression following transfection. The capability of cells to proliferate and differentiate into three major lineages (chondrocytes, adipocytes, and osteocytes) was found to be independent of the technique used for transfection. These results show that the microporation technique is superior to the others in terms of its ability to transfect hAD-MSCs without affecting their proliferation and differentiation capabilities. Therefore, this study provides a foundation for the selection of techniques when using ex vivo gene manipulation for cell-based gene therapy with MSCs as the vehicle for gene delivery.

Minicircle microporation-based non-viral gene delivery improved the targeting of mesenchymal stem cells to an injury site

“…Especially, Abdul Halim et al compared the transfection efficiencies of several methods (calcium phosphate precipitation, cationic liposome, electroporation, and microporation) in hAT-MSCs and found that only microporation generated 50% GFP þ MSCs whereas with the others made less than 1% of MSCs were produced in the display of GFP fluorescence [23], which is in accord with our result whereby microporation was much more efficient for MSC transfection than the cationic liposome method (Fig. 3).…”

“…2). Many studies have already reported that MSCs are resistant to plasmid transfection by the cationic liposome method [21,23,38,39]. In the case of minicircle, the cationic liposome method also seemed to be also inefficient although it showed higher transfection efficiency compared with only plasmid.…”

Substrate-mediated reprogramming of human fibroblasts into neural crest stem-like cells and their applications in neural repair

“…There are other more frequent methods to introduce foreign DNA into the nucleus of a cell, such as physical treatment (electroporation) [55], chemical carriers (cationic lipids) [56], or biological particles (viruses) as carriers [10]. These methods, especially for the viral carriers, have good transfection efficiency.…”

Emerging potential of gene silencing approaches targeting anti-chondrogenic factors for cell-based cartilage repair

“…In addition, Emerging potential of gene silencing approaches targeting anti-chondrogenic factors for cell-… this choice may be convenient for engineered cells to prime the regeneration process by triggering repair mechanisms as well as to stimulate an active involvement of the host tissues in the repair of the defect [30]. Transient techniques for gene silencing are receiving an increasing amount of interest, since the progress in RNAi techniques has led to the development and optimization of cell transfection in 3D microenvironment, with exogenous scaffolds being able to greatly increase efficiency and durability of gene silencing [29].…”