Simple, Efficient, and Reproducible Gene Transfection of Mouse Embryonic Stem Cells by Magnetofection

Abstract

Embryonic stem (ES) cells are recognized as an excellent cell culture model for studying developmental mechanisms and their therapeutic modulations. The aim of this work was to define whether using magnetofection was an efficient way to manipulate stem cells genetically without adversely affecting their proliferation or self-renewal capacity. We compared our magnetofection results to those of a conservative method using FuGENE 6. Using enhanced green fluorescent protein (eGFP) as a reporter gene in D3 mouse ES (mES) cells, we found that magnetofection gave a significantly higher efficiency (45%) of gene delivery in stem cells than did the FuGENE 6 method (15%), whereas both demonstrated efficient transfection in NIH-3T3 cells (60%). Although the transfected D3 (D3-eGFP) mES cells had undergone a large number of passages (>50), a high percentage of cells retained ES markers such as Oct-4 and stage-specific embryonic antigen-1 (SSEA-1). They also retained the ability to form embryoid bodies and differentiated in vitro into cells of the three germ layers. eGFP expression was sustained during stem cell proliferation and differentiation. This is the first transfection report using magnetofection in ES cells. On the basis of our results, we conclude that magnetofection is an efficient and reliable method for the introduction of foreign DNA into mouse ES cells and may become the method of choice.

Differentiation and Transplantation of Functional Pancreatic Beta Cells Generated from Induced Pluripotent Stem Cells Derived from a Type 1 Diabetes Mouse Model

“…For generation of exogenous DNA-free iPS cells, magnetbased nanofection was conducted according to the previous reports [17,36]. Briefly, the plasmid DNAs containing Octamer-binding transcription factor 4 (OCT4), SRY-box containing gene 2 (SOX2), Krü ppel-like factor 4 (KLF4), and c-MYC were mixed with PolyMAG (chemicell, GmbH, Berlin, Germany).…”

“…Such an autologous approach would eliminate the possibility of alloimmune rejection of transplanted cells. We propose that a combination of cellular reprogramming and differentiation techniques might be used for the generation of patient-specific iPSCs and their differentiation into pancreatic beta-like cells, and that such cells might provide a promising resource for cell therapy to treat diabetes without requiring high doses of immune-suppressive drugs [17][18][19].…”

Application of Magnet-based Nanofection in Embryonic Stem Cell Research

“…Magnet-based nanofection is a gene delivery system that utilizes nanotechnology. It is achieved by the application of a magnetic field to superparamagnetic iron peroxide particles which are associated with gene vectors (Lee et al, 2008). In this technique, the cationic polymer polyethyleneimine (PEI) is coated with superparamagnetic nanoparticles (tsMAG-PEI) are complexed to plasmid DNA.…”

“…Although magnet-based nanofection was studied in various cell lines, Lee et al originally used this technology for gene delivery in embryonic stem cells in 2008 (Lee et al, 2008). In this method, superparamagnetic nanoparticles (tsMAG) coated with cationic polymer polyethyleneimine (tsMAG-PEI) were combined with plasmid DNA.…”

“…Recently, it has been reported that magnet-based nanofection gave a significantly higher efficiency (45 %) of gene delivery in stem cells than did the FuGENE 6-mediated transfection method (15 %) and found that these cells maintained their usual un-differentiated characteristics of self-renewal and pluripotency for a long time (> 50 passages) (Lee et al, 2008). …”