Abstract
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 in vitro/in vivo 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.
Highlights
Induced pluripotent stem cells resemble embryonic stem (ES) cells in morphology, gene expression, epigenetic status, and in vitro differentiation [1,2]
The reprogramming efficiency of each treatment group was 0.032– 0.04% based on Alkaline phosphatase (AP)-positive clones, which constituted the input cells (1LMF-o: 0.04%, 1LMF-h: 0.035%, 2LMF-o: 0.032%, 2LMF-h: 0.039%), and there were no differences among the treatment groups
We introduced the liposomal magnetofection (LMF) method in order to enhance the reprogramming efficiency of somatic cells using non-viral vectors
Summary
Induced pluripotent stem (iPS) cells resemble embryonic stem (ES) cells in morphology, gene expression, epigenetic status, and in vitro differentiation [1,2]. While the excisable vector system yields a higher reprogramming efficiency (.100-fold) than other non-viral systems, laborious screening of excised lines and examination of non-specific genetic alteration is inevitably required before and after transfection. Nanotechnologies have shown great potential to enhance stem-cell research and stem-cell–based therapeutics. Such methods could be useful in measuring, understanding, and manipulating stem cells [20]. As a universal method enhancing non-viral gene delivery, magnetofection (MF) can be an efficient and reliable method for the introduction of foreign DNA into target cells. In the case of iPS cell generation, we expect that the efficiency of non-viral gene delivery can be increased by MF using nanoparticles or polyplexes
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