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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

MNP-mediated delivery of a functional gene encoding FGF2—effect of magnetofection on transfection efficiency. Monolayers (n = 3 cultures) were transfected with Neuromag complexed with either pFGF2-GFP, pAN-GFP (control plasmid lacking the FGF2 insert) or pmaxGFP (positive control), with application of the indicated magnetic fields, then studied at 48 h post-transfection. (A) Representative phase and fluorescence double-merged image of cells transfected with pFGF2-GFP, demonstrating nuclear expression of GFP. Inset is a representative image of cells transfected with pAN-GFP; note that GFP expression extends throughout the cytoplasm. (B) Bar chart showing the proportions of transfected NSCs under no magnetic field (none), static magnetic field (F0) and oscillating magnetic field (F = 4 Hz; F4) conditions. *P < 0.05 and ***P < 0.001 for inter-field comparisons (indicated at top of chart) for a given plasmid; +++P < 0.001 versus pmaxGFP for a given magnetic field condition (one-way ANOVA and Bonferroni’s MCT); n = 3 cultures. (C) Regression analysis demonstrating transfection efficiency is inversely related to plasmid size under no magnetic field (None; r2 = 0.994; P < 0.05), static magnetic field (F0; r2 = 0.998; P < 0.05) and oscillating (F = 4 Hz) magnetic field (F4; r2 = 0.999; P < 0.01) conditions. (D) Immunoblots sequentially probed with antibodies to FGF2 (top) and β-actin (loading control; bottom), demonstrating expression of a 60 kDa protein species (indicated by arrow) in extracts of cells (n = 3 cultures) transfected with pFGF2-GFP (lanes 2, 4 and 6) but not with pAN-GFP (lanes 1, 3 and 5); the migration of size markers is displayed on the right-hand side. Scale bar = 5 µm in (A).
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jfb-06-00259-f004: MNP-mediated delivery of a functional gene encoding FGF2—effect of magnetofection on transfection efficiency. Monolayers (n = 3 cultures) were transfected with Neuromag complexed with either pFGF2-GFP, pAN-GFP (control plasmid lacking the FGF2 insert) or pmaxGFP (positive control), with application of the indicated magnetic fields, then studied at 48 h post-transfection. (A) Representative phase and fluorescence double-merged image of cells transfected with pFGF2-GFP, demonstrating nuclear expression of GFP. Inset is a representative image of cells transfected with pAN-GFP; note that GFP expression extends throughout the cytoplasm. (B) Bar chart showing the proportions of transfected NSCs under no magnetic field (none), static magnetic field (F0) and oscillating magnetic field (F = 4 Hz; F4) conditions. *P < 0.05 and ***P < 0.001 for inter-field comparisons (indicated at top of chart) for a given plasmid; +++P < 0.001 versus pmaxGFP for a given magnetic field condition (one-way ANOVA and Bonferroni’s MCT); n = 3 cultures. (C) Regression analysis demonstrating transfection efficiency is inversely related to plasmid size under no magnetic field (None; r2 = 0.994; P < 0.05), static magnetic field (F0; r2 = 0.998; P < 0.05) and oscillating (F = 4 Hz) magnetic field (F4; r2 = 0.999; P < 0.01) conditions. (D) Immunoblots sequentially probed with antibodies to FGF2 (top) and β-actin (loading control; bottom), demonstrating expression of a 60 kDa protein species (indicated by arrow) in extracts of cells (n = 3 cultures) transfected with pFGF2-GFP (lanes 2, 4 and 6) but not with pAN-GFP (lanes 1, 3 and 5); the migration of size markers is displayed on the right-hand side. Scale bar = 5 µm in (A).

Mentions: Cells which had been transfected with pFGF2-GFP displayed a characteristic pattern of predominantly nuclear GFP expression (Figure 4A; main image) irrespective of the magnetic field condition. This contrasted with the more extensive cellular distribution of GFP after transfection with either pAN-GFP (Figure 4A inset) or pmaxGFP (e.g., see Figure 1B). The application of magnetic fields enhanced the transfection of all three plasmids; an oscillating field of 4 Hz yielded higher transfection efficiencies than a static field in all cases (Figure 4B). However, under each magnetic field condition, the proportions of GFP-expressing cells were lower after transfection with either pFGF2-GFP or pAN-GFP than with pmaxGFP. Notably, transfection efficiency was inversely related to plasmid size for all magnetic fields (Figure 4C).


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 delivery of a functional gene encoding FGF2—effect of magnetofection on transfection efficiency. Monolayers (n = 3 cultures) were transfected with Neuromag complexed with either pFGF2-GFP, pAN-GFP (control plasmid lacking the FGF2 insert) or pmaxGFP (positive control), with application of the indicated magnetic fields, then studied at 48 h post-transfection. (A) Representative phase and fluorescence double-merged image of cells transfected with pFGF2-GFP, demonstrating nuclear expression of GFP. Inset is a representative image of cells transfected with pAN-GFP; note that GFP expression extends throughout the cytoplasm. (B) Bar chart showing the proportions of transfected NSCs under no magnetic field (none), static magnetic field (F0) and oscillating magnetic field (F = 4 Hz; F4) conditions. *P < 0.05 and ***P < 0.001 for inter-field comparisons (indicated at top of chart) for a given plasmid; +++P < 0.001 versus pmaxGFP for a given magnetic field condition (one-way ANOVA and Bonferroni’s MCT); n = 3 cultures. (C) Regression analysis demonstrating transfection efficiency is inversely related to plasmid size under no magnetic field (None; r2 = 0.994; P < 0.05), static magnetic field (F0; r2 = 0.998; P < 0.05) and oscillating (F = 4 Hz) magnetic field (F4; r2 = 0.999; P < 0.01) conditions. (D) Immunoblots sequentially probed with antibodies to FGF2 (top) and β-actin (loading control; bottom), demonstrating expression of a 60 kDa protein species (indicated by arrow) in extracts of cells (n = 3 cultures) transfected with pFGF2-GFP (lanes 2, 4 and 6) but not with pAN-GFP (lanes 1, 3 and 5); the migration of size markers is displayed on the right-hand side. Scale bar = 5 µm in (A).
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Related In: Results  -  Collection

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jfb-06-00259-f004: MNP-mediated delivery of a functional gene encoding FGF2—effect of magnetofection on transfection efficiency. Monolayers (n = 3 cultures) were transfected with Neuromag complexed with either pFGF2-GFP, pAN-GFP (control plasmid lacking the FGF2 insert) or pmaxGFP (positive control), with application of the indicated magnetic fields, then studied at 48 h post-transfection. (A) Representative phase and fluorescence double-merged image of cells transfected with pFGF2-GFP, demonstrating nuclear expression of GFP. Inset is a representative image of cells transfected with pAN-GFP; note that GFP expression extends throughout the cytoplasm. (B) Bar chart showing the proportions of transfected NSCs under no magnetic field (none), static magnetic field (F0) and oscillating magnetic field (F = 4 Hz; F4) conditions. *P < 0.05 and ***P < 0.001 for inter-field comparisons (indicated at top of chart) for a given plasmid; +++P < 0.001 versus pmaxGFP for a given magnetic field condition (one-way ANOVA and Bonferroni’s MCT); n = 3 cultures. (C) Regression analysis demonstrating transfection efficiency is inversely related to plasmid size under no magnetic field (None; r2 = 0.994; P < 0.05), static magnetic field (F0; r2 = 0.998; P < 0.05) and oscillating (F = 4 Hz) magnetic field (F4; r2 = 0.999; P < 0.01) conditions. (D) Immunoblots sequentially probed with antibodies to FGF2 (top) and β-actin (loading control; bottom), demonstrating expression of a 60 kDa protein species (indicated by arrow) in extracts of cells (n = 3 cultures) transfected with pFGF2-GFP (lanes 2, 4 and 6) but not with pAN-GFP (lanes 1, 3 and 5); the migration of size markers is displayed on the right-hand side. Scale bar = 5 µm in (A).
Mentions: Cells which had been transfected with pFGF2-GFP displayed a characteristic pattern of predominantly nuclear GFP expression (Figure 4A; main image) irrespective of the magnetic field condition. This contrasted with the more extensive cellular distribution of GFP after transfection with either pAN-GFP (Figure 4A inset) or pmaxGFP (e.g., see Figure 1B). The application of magnetic fields enhanced the transfection of all three plasmids; an oscillating field of 4 Hz yielded higher transfection efficiencies than a static field in all cases (Figure 4B). However, under each magnetic field condition, the proportions of GFP-expressing cells were lower after transfection with either pFGF2-GFP or pAN-GFP than with pmaxGFP. Notably, transfection efficiency was inversely related to plasmid size for all magnetic fields (Figure 4C).

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