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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.


MNP–mediated combinatorial gene delivery to NSC monolayers. Cultures (n = 3) were magnetofected (oscillating magnetic field of F = 4 Hz) with complexes formed between Neuromag MNPs and either pDRE2, pmaxGFP or pDRE2 plus pmaxGFP (1:1 mix) plasmids; in all transfections, the final concentration of each plasmid was half that employed in the standard protocol. (A) Representative image of cells co-transfected with both plasmids. (A, insets) same field of cells in (A), showing GFP or RFP expression alone at 48 h post-transfection; (B) Bar chart showing the proportions of transfected cells that express GFP plus RFP, GFP alone and RFP alone after co-transfection of plasmids; (C) Bar chart showing transfection efficiencies for co-transfection and the corresponding single gene transfection controls. Scale bar = 20 µm in (A–C).
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jfb-06-00259-f003: MNP–mediated combinatorial gene delivery to NSC monolayers. Cultures (n = 3) were magnetofected (oscillating magnetic field of F = 4 Hz) with complexes formed between Neuromag MNPs and either pDRE2, pmaxGFP or pDRE2 plus pmaxGFP (1:1 mix) plasmids; in all transfections, the final concentration of each plasmid was half that employed in the standard protocol. (A) Representative image of cells co-transfected with both plasmids. (A, insets) same field of cells in (A), showing GFP or RFP expression alone at 48 h post-transfection; (B) Bar chart showing the proportions of transfected cells that express GFP plus RFP, GFP alone and RFP alone after co-transfection of plasmids; (C) Bar chart showing transfection efficiencies for co-transfection and the corresponding single gene transfection controls. Scale bar = 20 µm in (A–C).

Mentions: Given the complex nature of neural pathologies, it is unlikely that delivery of a single gene will be sufficient to augment regenerative processes in areas of neural injury, consequently combinatorial gene delivery was assessed here to more rigorously assess the clinical translational potential of the developed protocol. In co-transfected cultures, expression of both RFP and GFP could be clearly observed (Figure 3A main image and insets), with the majority of transfected cells expressing both reporter proteins. In all cases, co-transfected cells expressed normal cellular and nuclear morphologies with no evidence of cell rounding or loss, suggesting that combinatorial delivery is safe. On average, 87% of transfected cells expressed RFP plus GFP, whilst 11% expressed GFP only and the remaining 2% expressed RFP only (Figure 3B). These findings were in accordance with the results of the single plasmid transfection controls, which demonstrated a tendency (P = 0.056; paired Students t-test; n = 3 cultures) towards a lower transfection efficacy for the RFP-encoding plasmid versus the GFP-encoding plasmid (21.5% ± 1.9% versus 29.3% ± 2.8%) (Figure 3C). This is in accordance with the observation that transfection efficiency declines with increasing plasmid size (see Section 2.4), since the RFP-encoding plasmid is larger than the GFP-encoding plasmid (4.6 kb versus 3.5 kb).


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)

MNP–mediated combinatorial gene delivery to NSC monolayers. Cultures (n = 3) were magnetofected (oscillating magnetic field of F = 4 Hz) with complexes formed between Neuromag MNPs and either pDRE2, pmaxGFP or pDRE2 plus pmaxGFP (1:1 mix) plasmids; in all transfections, the final concentration of each plasmid was half that employed in the standard protocol. (A) Representative image of cells co-transfected with both plasmids. (A, insets) same field of cells in (A), showing GFP or RFP expression alone at 48 h post-transfection; (B) Bar chart showing the proportions of transfected cells that express GFP plus RFP, GFP alone and RFP alone after co-transfection of plasmids; (C) Bar chart showing transfection efficiencies for co-transfection and the corresponding single gene transfection controls. Scale bar = 20 µm in (A–C).
© Copyright Policy
Related In: Results  -  Collection

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

jfb-06-00259-f003: MNP–mediated combinatorial gene delivery to NSC monolayers. Cultures (n = 3) were magnetofected (oscillating magnetic field of F = 4 Hz) with complexes formed between Neuromag MNPs and either pDRE2, pmaxGFP or pDRE2 plus pmaxGFP (1:1 mix) plasmids; in all transfections, the final concentration of each plasmid was half that employed in the standard protocol. (A) Representative image of cells co-transfected with both plasmids. (A, insets) same field of cells in (A), showing GFP or RFP expression alone at 48 h post-transfection; (B) Bar chart showing the proportions of transfected cells that express GFP plus RFP, GFP alone and RFP alone after co-transfection of plasmids; (C) Bar chart showing transfection efficiencies for co-transfection and the corresponding single gene transfection controls. Scale bar = 20 µm in (A–C).
Mentions: Given the complex nature of neural pathologies, it is unlikely that delivery of a single gene will be sufficient to augment regenerative processes in areas of neural injury, consequently combinatorial gene delivery was assessed here to more rigorously assess the clinical translational potential of the developed protocol. In co-transfected cultures, expression of both RFP and GFP could be clearly observed (Figure 3A main image and insets), with the majority of transfected cells expressing both reporter proteins. In all cases, co-transfected cells expressed normal cellular and nuclear morphologies with no evidence of cell rounding or loss, suggesting that combinatorial delivery is safe. On average, 87% of transfected cells expressed RFP plus GFP, whilst 11% expressed GFP only and the remaining 2% expressed RFP only (Figure 3B). These findings were in accordance with the results of the single plasmid transfection controls, which demonstrated a tendency (P = 0.056; paired Students t-test; n = 3 cultures) towards a lower transfection efficacy for the RFP-encoding plasmid versus the GFP-encoding plasmid (21.5% ± 1.9% versus 29.3% ± 2.8%) (Figure 3C). This is in accordance with the observation that transfection efficiency declines with increasing plasmid size (see Section 2.4), since the RFP-encoding plasmid is larger than the GFP-encoding plasmid (4.6 kb versus 3.5 kb).

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.