(584ab) Nanog Transient Overexpression With Optimized Magnitofection to Reverse the Effects of Organismal Aging On Mesenchymal Stem Cells

Introduction: Previously, our group demonstrated that overexpression of a single transcription factor, Nanog, can reverse the effects of organismal aging on mesenchymal stem cells (MSCs) proliferation and myogenic differentiation [1]. We also found that transient Nanog overexpression is sufficient to reverse the effects of aging, thereby suggesting the permanent genetic modification may not be necessary. This result prompted us to hypothesize that transient overexpression of Nanog in MSCs by DNA transfection may overcome the effects of cellular senescence. However, it has been proven difficult to deliver genes into MSCs. We hypothesized that we could have efficient gene delivery to achieve our goal by using DNA magnetofection.

Materials and Methods: Human hair follicle derived MSCs (HFMSCs) were used in this study. EGFP- or Nanog-encoding plasmid first formed complexes with polyMAG nanoparticles and pulled down by magnetic field to the cells seeded in 24-well plates. Chloroquine was used to enhance endosomal escape of internalized DNA complexes.

Results and Discussion: Magnetofection on HFMSCs was first optimized with EGFP-expressing plasmid. The optimized dose for transfection without detrimental effects on cell viability was 0.5μgDNA/0.5μLparticles complex. The green positive (G+) cells reached 17.46%±1.21 and the total green fluoresce intensity (GFI) increased by 1.98±0.06 fold comparing to control cells. Repeated addition of optimal magnetofection complex for tree time enhanced the transfection efficiency to G+=47.06%±2.25 (GFI increased by 3.09±0.08 fold) and treatment with choloroquine further promoted the transfection efficiency to 73.64%±2.57; (GFI= 5.19±0.22 fold). This approach is significantly more efficient than the widely used transfection reagent, Lipofectamine 2000, which showed only G+:15.0%±0.51and GFI=2.58±0.07 fold at its optimal DNA/reagent ratio and with lower cell viability. This optimized protocol is currently applied to other MSC such as bone marrow (BM)-MSC as well as for delivery Nanog-expressing plasmid to evaluate the effects of gene transfer on cellular senescence. 

Conclusion: This study provides a possible alternative, DNA magnetofection, to overcome the difficulty of DNA delivery to MSCs. Our results imply that this strategy may be employed to reverse the effects of organismal aging or culture senescence on MSC without viral transduction and permanent genome modification.

[1] Han J, Mistriotis P, Lei P, Wang D, Liu S, Andreadis ST. Nanog reverses the effects of organismal aging on mesenchymal stem cell proliferation and myogenic differentiation potential. Stem Cells. 2012;30:2746-59.