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Using magnetic nanoparticles for gene transfer to neural stem cells: stem cell propagation method influences outcomes.

Pickard MR, Adams CF, Barraud P, Chari DM - J Funct Biomater (2015)

Bottom Line: 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).MNPs deployed with oscillating magnetic fields ("magnetofection technology") mediate effective gene transfer to neurospheres but the efficacy of this approach for monolayers is unknown.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.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Neural Engineering Group, Institute for Science and Technology in Medicine, Keele University, Keele, Staffordshire ST5 5BG, UK. m.r.pickard@keele.ac.uk.

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.

No MeSH data available.


Related in: MedlinePlus

Effect of magnetofection with static (F = 0 Hz) and oscillating (F = 0.5–4 Hz) magnetic fields on transfection efficiency. (A) Representative phase image of monolayer cultures; (B) Representative double-merged image of DAPI-stained cultures at 48 h after Neuromag-mediated transfection with pmaxGFP conducted in the absence of a magnetic field; (C) Representative double-merged image of DAPI-stained cultures at 48 h after Neuromag-mediated transfection with pmaxGFP with an applied oscillating magnetic field of F = 4 Hz; (D) Bar chart showing proportions of transfected cells in NSC monolayers at 48 h after addition of Neuromag and pmaxGFP complexes with application of the indicated magnetic field.*P < 0.05 & ***P < 0.001 versus no magnetic field; +++P < 0.001 versus static (F = 0 Hz) magnetic field; n = 4 cultures (one-way ANOVA and Bonferroni’s MCT). Scale bar = 200 µm in (A, B & C).
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jfb-06-00259-f001: Effect of magnetofection with static (F = 0 Hz) and oscillating (F = 0.5–4 Hz) magnetic fields on transfection efficiency. (A) Representative phase image of monolayer cultures; (B) Representative double-merged image of DAPI-stained cultures at 48 h after Neuromag-mediated transfection with pmaxGFP conducted in the absence of a magnetic field; (C) Representative double-merged image of DAPI-stained cultures at 48 h after Neuromag-mediated transfection with pmaxGFP with an applied oscillating magnetic field of F = 4 Hz; (D) Bar chart showing proportions of transfected cells in NSC monolayers at 48 h after addition of Neuromag and pmaxGFP complexes with application of the indicated magnetic field.*P < 0.05 & ***P < 0.001 versus no magnetic field; +++P < 0.001 versus static (F = 0 Hz) magnetic field; n = 4 cultures (one-way ANOVA and Bonferroni’s MCT). Scale bar = 200 µm in (A, B & C).

Mentions: Healthy monolayer cultures of NSCs were routinely derived from dissociated neurospheres, predominantly comprising cells with elongated cell bodies with two or more processes (Figure 1A). In preliminary transfection experiments (n = 2 cultures) using Neuromag at 2 µL/well or 6.9 µL/mL culture medium (i.e., a concentration recommended by Oz Biosciences for neuronal transfection), marked toxicity was observed in monolayers with an applied static magnetic field, evidenced by reduced cell adherence and rounding at 48 h (data not shown); this was not apparent in the absence of a magnetic field. Under an applied static magnetic field, negligible cytotoxicity was observed when the Neuromag dose was reduced to 0.62 µL/well or 2.1 µL/mL culture medium (data not shown). As procedural safety was of paramount concern in these experiments (to develop a method that is suitable for clinical translation), the latter dose was employed in all further monolayer experiments. For the same reason, a positive control, for example, a common non-viral procedure such as nucleofection was not studied here as this can result in significant loss of cell viability despite high transfection efficiency [15].


Using magnetic nanoparticles for gene transfer to neural stem cells: stem cell propagation method influences outcomes.

Pickard MR, Adams CF, Barraud P, Chari DM - J Funct Biomater (2015)

Effect of magnetofection with static (F = 0 Hz) and oscillating (F = 0.5–4 Hz) magnetic fields on transfection efficiency. (A) Representative phase image of monolayer cultures; (B) Representative double-merged image of DAPI-stained cultures at 48 h after Neuromag-mediated transfection with pmaxGFP conducted in the absence of a magnetic field; (C) Representative double-merged image of DAPI-stained cultures at 48 h after Neuromag-mediated transfection with pmaxGFP with an applied oscillating magnetic field of F = 4 Hz; (D) Bar chart showing proportions of transfected cells in NSC monolayers at 48 h after addition of Neuromag and pmaxGFP complexes with application of the indicated magnetic field.*P < 0.05 & ***P < 0.001 versus no magnetic field; +++P < 0.001 versus static (F = 0 Hz) magnetic field; n = 4 cultures (one-way ANOVA and Bonferroni’s MCT). Scale bar = 200 µm in (A, B & C).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4493511&req=5

jfb-06-00259-f001: Effect of magnetofection with static (F = 0 Hz) and oscillating (F = 0.5–4 Hz) magnetic fields on transfection efficiency. (A) Representative phase image of monolayer cultures; (B) Representative double-merged image of DAPI-stained cultures at 48 h after Neuromag-mediated transfection with pmaxGFP conducted in the absence of a magnetic field; (C) Representative double-merged image of DAPI-stained cultures at 48 h after Neuromag-mediated transfection with pmaxGFP with an applied oscillating magnetic field of F = 4 Hz; (D) Bar chart showing proportions of transfected cells in NSC monolayers at 48 h after addition of Neuromag and pmaxGFP complexes with application of the indicated magnetic field.*P < 0.05 & ***P < 0.001 versus no magnetic field; +++P < 0.001 versus static (F = 0 Hz) magnetic field; n = 4 cultures (one-way ANOVA and Bonferroni’s MCT). Scale bar = 200 µm in (A, B & C).
Mentions: Healthy monolayer cultures of NSCs were routinely derived from dissociated neurospheres, predominantly comprising cells with elongated cell bodies with two or more processes (Figure 1A). In preliminary transfection experiments (n = 2 cultures) using Neuromag at 2 µL/well or 6.9 µL/mL culture medium (i.e., a concentration recommended by Oz Biosciences for neuronal transfection), marked toxicity was observed in monolayers with an applied static magnetic field, evidenced by reduced cell adherence and rounding at 48 h (data not shown); this was not apparent in the absence of a magnetic field. Under an applied static magnetic field, negligible cytotoxicity was observed when the Neuromag dose was reduced to 0.62 µL/well or 2.1 µL/mL culture medium (data not shown). As procedural safety was of paramount concern in these experiments (to develop a method that is suitable for clinical translation), the latter dose was employed in all further monolayer experiments. For the same reason, a positive control, for example, a common non-viral procedure such as nucleofection was not studied here as this can result in significant loss of cell viability despite high transfection efficiency [15].

Bottom Line: 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).MNPs deployed with oscillating magnetic fields ("magnetofection technology") mediate effective gene transfer to neurospheres but the efficacy of this approach for monolayers is unknown.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.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Neural Engineering Group, Institute for Science and Technology in Medicine, Keele University, Keele, Staffordshire ST5 5BG, UK. m.r.pickard@keele.ac.uk.

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.

No MeSH data available.


Related in: MedlinePlus