Efficient Generation of Virus-Free iPS Cells Using Liposomal Magnetofection

<div><p>The generation of induced pluripotent stem (iPS) cells is a powerful tool in regenerative medicine, and advances in nanotechnology clearly have great potential to enhance stem cell research. Here, we introduce a liposomal magnetofection (LMF) method for iPS cell generation. Efficient conditions for generating virus-free iPS cells from mouse embryonic fibroblast (MEF) cells were determined through the use of different concentrations of CombiMag nanoparticle-DNA(pCX-OKS-2A and pCX-cMyc)-lipoplexes and either one or two cycles of the LMF procedure. The cells were prepared in a short reprogramming time period (≤8 days, 0.032–0.040%). Among the seven LMF-iPS cell lines examined, two were confirmed to be integration-free, and an integration-free LMF-iPS cell line was produced under the least toxic conditions (single LMF cycle with a half-dose of plasmid). This cell line also displayed <i>in vitro</i>/<i>in vivo</i> pluripotency, including teratoma formation and chimeric mouse production. In addition, the safety of CombiMag-DNA lipoplexes for the transfection of MEF cells was confirmed through lactate dehydrogenase activity assay and transmission electron microscopy. These results demonstrated that the LMF method is simple, effective, and safe. LMF may represent a superior technique for the generation of virus-free or integration-free iPS cell lines that could lead to enhanced stem cell therapy in the future.</p></div

Comparison of <i>in vitro</i> differentiation potential between integrated (1LMF-h-iPS1) and integration-free LMF-iPS cell lines (1LMF-h-iPS2).

<p>(A) Phase-contrast images of the two LMF-iPS cell lines, according to spontaneous differentiation for 20 days. (B) RT-PCR analysis of the spontaneous differentiation of the two LMF-iPS cell lines into three germ-like layers expressing undifferentiated ES cell markers (Oct4 and Nanog), as well as endodermal (α-fetoprotein and α-amylase), mesodermal (β-enolase and renin), and ectodermal (Map2 and β-tubulin) genes. (C) Immunocytochemistry of neuronal cell differentiation in the two LMF-iPS cell lines into Map2-, Tuj1-, and GFAP-positive cells. (D and E) RT-PCR and immunostaining results showing cardiac (TnI) and endothelial (Tie2) cell differentiation of two LMF-iPS cell lines. Blue nuclear staining is by DAPI.</p

<i>In vivo</i> validation of the pluripotency of 1LMF-h-iPS 1 and 1LMF-h-iPS 2 cells.

<p>Teratoma formation in mice and tissue sections showing the formation of cells from the three germ layers: endoderm (gut epithelium and secretory epithelium), mesoderm (adipose, bone, cartilage, and striated muscle fibers), and ectoderm (epidermis and neural rosettes) in 1LMF-h-iPS 1 (A) and 1LMF-h-iPS 2 cells (B). (C and C’) Chimeric mouse produced by injecting 1LMF-h-iPS 2 cells into c57BL/6 host blastocysts. The natural coat color of the surrogate strain ICR is white, and the brown hair (red arrowhead) indicates a chimeric mouse (red) that originated from the 1LMF-h-iPS 2 cells.</p

Cytotoxicity determination of MEF cells after LMF.

<p>(A) Liposomal magnetofection particles (arrows) in MEF cells at 24 hr post-LMF with either one or two LMF procedures and with different DNA doses (o: 1.5 µg, h: 0.75 µg). (B and C) The colorimetric absorbance values and LDH release percentages for control MEF cells, the four LMF treatment groups (1LMF-o, 1LMF-h, 2LMF-o, and 2LMF-h) at 24 hr post-LMF, and triton X-100–exposed MEF cells.</p

Generation of mouse induced pluripotent stem cells by liposomal magnetofection (LMF) under different conditions.

<p>Generation of mouse induced pluripotent stem cells by liposomal magnetofection (LMF) under different conditions.</p

Characterization of LMF-iPS cells: comparison of MEF and mouse D3 ES cells.

<p>(A) Alkaline phosphatase (AP) staining. Seven LMF-iPS cell lines examined expressed high levels of AP, similar to D3 ES cells. (B) Immunofluorescence staining of the ES cell-specific markers Oct4 and stage-specific embryonic antigen 1 (SSEA1). Scale bars: 200 µm. (C) mRNA expression of ES cell-specific markers (Nanog, Tert, Zfp, Oct4, and Sox2) and exogenous genes (Kl4 and c-Myc). G3PDH was used as a loading control.</p

Transmission electron microscopic analysis of MEFs, CombiMag-DNA lipoplexes, LMF-treated MEF cells at 12 hr intervals until 48 hrs, and two LMF-iPS cell lines.

<p>The lower photos represent magnifications of the white-dotted square in each upper photo, and the white arrowheads indicate the presence of liposomal magnetofection particles in LMF-MEF cells. Upper: low magnification, x6,000–8,000; lower: high magnification, x50,000.</p

Generation of iPS cells from mouse embryonic fibroblasts (MEF) by liposomal magnetofection (LMF) of two plasmids (pCX-OKS-2A and pCX-cMyc).

<p>(A) Methodology of LMF. Two plasmids were mixed with liposomes for 15 min and the resulting DNA lipoplexes were then mixed with CombiMag nanoparticles for a further 15 min. The CombiMag-DNA lipoplexes were added into MEF culture dishes, which were then placed on a magnetic plate. (B) Timeline of LMF-iPS cell production with either one cycle of LMF (1LMF, upper) or two cycles of LMF (2LMF, lower). (C) Morphological changes of MEF cells after LMF and culture of LMF-iPS cells. Two days after LMF, MEF cells were morphologically transformed, and small aggregates were observed at 5–6 days. ES cell-like LMF-iPS cell colonies appeared at 8 days. These colonies exhibited strong alkaline phosphatase (AP) expression. Four passage-cultured feeder-dependent and feeder-independent LMF-iPS cell colonies are shown, with morphological similarity to typical ES cells. Scale bars, 100 µm.</p