Using Magnetic Nanoparticles for Gene Transfer to Neural Stem Cells: Stem Cell Propagation Method Influences Outcomes

Abstract

Genetically engineered neural stem cell (NSC) transplants offer a key strategy to augment neural repair by releasing therapeutic biomolecules into injury sites. Genetic modification of NSCs is heavily reliant on viral vectors but cytotoxic effects have prompted development of non-viral alternatives, such as magnetic nanoparticle (MNPs). NSCs are propagated in laboratories as either 3-D suspension “neurospheres” or 2-D adherent “monolayers”. MNPs deployed with oscillating magnetic fields (“magnetofection technology”) mediate effective gene transfer to neurospheres but the efficacy of this approach for monolayers is unknown. It is important to address this issue as oscillating magnetic fields dramatically enhance MNP-based transfection in transplant cells (e.g., astrocytes and oligodendrocyte precursors) propagated as monolayers. We report for the first time that oscillating magnetic fields enhanced MNP-based transfection with reporter and functional (basic fibroblast growth factor; FGF2) genes in monolayer cultures yielding high transfection versus neurospheres. Transfected NSCs showed high viability and could re-form neurospheres, which is important as neurospheres yield higher post-transplantation viability versus monolayer cells. Our results demonstrate that the combination of oscillating magnetic fields and a monolayer format yields the highest efficacy for MNP-mediated gene transfer to NSCs, offering a viable non-viral alternative for genetic modification of this important neural cell transplant population.

Part I: Minicircle vector technology limits DNA size restrictions on ex vivo gene delivery using nanoparticle vectors: Overcoming a translational barrier in neural stem cell therapy

“…We recently reported technically simple, rapid and safe magnetofection protocols for genetic modification of major neural transplant types including NSCs, with oscillating magnetic fields always outperforming static fields [21][22][23][24][25]. MNPs have emerged strongly as a class of advanced functional materials for neuro-regeneration, serving as 'multifunctional tools' for cell therapy given additional applications for non-invasive cell imaging and magnetic cell targeting, so it is clear that this non-viral gene transfer method offers significant benefits for clinical translation [26].…”

“…MNPs have emerged strongly as a class of advanced functional materials for neuro-regeneration, serving as 'multifunctional tools' for cell therapy given additional applications for non-invasive cell imaging and magnetic cell targeting, so it is clear that this non-viral gene transfer method offers significant benefits for clinical translation [26]. Despite the critical advantages offered by this technique, we and others have reported that DNA plasmid size bears an inverse relationship with transfection efficiency for non-viral methods such as liposome-and nanoparticle-mediated transfection [23,[27][28][29]. For example, using lipofection, a systematic analysis of luciferase expression levels (using reporter constructs of increasing size; 4.8 kb to 10.5 kb) showed reduced transfection with greater plasmid size [29].…”

“…Similarly, we reported an inverse relationship between transfection efficiency and plasmid size when using reporter plasmids (3.6 kb, transfection efficiency, ca. 32.2%) versus therapeutic constructs encoding basic fibroblast growth factor (7.4 kb, transfection efficiency: 13.5%) [23]. As therapeutic DNA constructs inevitably increase DNA size using conventional plasmids, this reduction in transfection efficiency and the functionality of nanoparticle vectors, is a critical translational barrier for ex vivo neural gene therapy.…”

“…Most non-viral studies employ DNA vectors expressing variants of GFP that provide enhanced levels of brightness such as TurboGFP and TagGFP [23,36], whereas in this study, pp-GFP and mC-GFP express a non-toxic, non-aggregating but less bright 'copGFP'. The In order to optimize protocols to achieve high transfection levels, a range of pp-GFP and mC-GFP copy numbers per well were assessed; the oscillation frequency previously determined to be optimal for NSC transfection (4Hz) was used for all experiments [23].…”

“…The In order to optimize protocols to achieve high transfection levels, a range of pp-GFP and mC-GFP copy numbers per well were assessed; the oscillation frequency previously determined to be optimal for NSC transfection (4Hz) was used for all experiments [23]. Transfection efficiency was overall lower in cells transfected with pp-GFP compared to mC-GFP, with the highest transfection efficiency observed when using a mC copy number of 6 x 10 11 .…”