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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: 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).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

Magnetofection of a plasmid encoding FGF2 has no effect on cell viability and stimulates cell proliferation in the absence of exogenous FGF2. Monolayers (n = 3 cultures) were transfected with Neuromag complexed with either pFGF2-GFP or pAN-GFP (control plasmid), with application of an oscillating (F = 4 Hz) magnetic field. At 48 h post-transfection, cells were detached, counted (to assess cytotoxicity) and allowed to form neurospheres in culture medium with/without exogenous FGF2. (A) Bar chart of total cell count and (B) cell viability prior to neurosphere formation; (C) Bar chart showing neurosphere number and (D) size at 96 h after plating in neurosphere medium ± exogenous FGF2. *P < 0.05, **P < 0.01 and ***P < 0.001 (one-way ANOVA and Bonferroni’s MCT). (E) Bar chart showing total cell count after dissociation of neurospheres at 144 h after plating in neurosphere medium ± exogenous FGF2. *P < 0.05, **P < 0.01 and ***P < 0.001 (one-way ANOVA and Bonferroni’s MCT).
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jfb-06-00259-f005: Magnetofection of a plasmid encoding FGF2 has no effect on cell viability and stimulates cell proliferation in the absence of exogenous FGF2. Monolayers (n = 3 cultures) were transfected with Neuromag complexed with either pFGF2-GFP or pAN-GFP (control plasmid), with application of an oscillating (F = 4 Hz) magnetic field. At 48 h post-transfection, cells were detached, counted (to assess cytotoxicity) and allowed to form neurospheres in culture medium with/without exogenous FGF2. (A) Bar chart of total cell count and (B) cell viability prior to neurosphere formation; (C) Bar chart showing neurosphere number and (D) size at 96 h after plating in neurosphere medium ± exogenous FGF2. *P < 0.05, **P < 0.01 and ***P < 0.001 (one-way ANOVA and Bonferroni’s MCT). (E) Bar chart showing total cell count after dissociation of neurospheres at 144 h after plating in neurosphere medium ± exogenous FGF2. *P < 0.05, **P < 0.01 and ***P < 0.001 (one-way ANOVA and Bonferroni’s MCT).

Mentions: In agreement with earlier findings, for all three plasmids, microscopy revealed no obvious adverse effects on cell morphology and cell adherence for cells treated with complexes compared with cells treated with plasmid alone. Further analysis of cells transfected under oscillating (F = 4 Hz) magnetic field conditions, i.e., conditions which yield the highest transfection efficiencies, revealed normal total counts and cell viability at 48 h after delivery of either pFGF2-GFP or pAN-GFP (Figure 5A,B), consistent with findings for pmaxGFP.


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)

Magnetofection of a plasmid encoding FGF2 has no effect on cell viability and stimulates cell proliferation in the absence of exogenous FGF2. Monolayers (n = 3 cultures) were transfected with Neuromag complexed with either pFGF2-GFP or pAN-GFP (control plasmid), with application of an oscillating (F = 4 Hz) magnetic field. At 48 h post-transfection, cells were detached, counted (to assess cytotoxicity) and allowed to form neurospheres in culture medium with/without exogenous FGF2. (A) Bar chart of total cell count and (B) cell viability prior to neurosphere formation; (C) Bar chart showing neurosphere number and (D) size at 96 h after plating in neurosphere medium ± exogenous FGF2. *P < 0.05, **P < 0.01 and ***P < 0.001 (one-way ANOVA and Bonferroni’s MCT). (E) Bar chart showing total cell count after dissociation of neurospheres at 144 h after plating in neurosphere medium ± exogenous FGF2. *P < 0.05, **P < 0.01 and ***P < 0.001 (one-way ANOVA and Bonferroni’s MCT).
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Related In: Results  -  Collection

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jfb-06-00259-f005: Magnetofection of a plasmid encoding FGF2 has no effect on cell viability and stimulates cell proliferation in the absence of exogenous FGF2. Monolayers (n = 3 cultures) were transfected with Neuromag complexed with either pFGF2-GFP or pAN-GFP (control plasmid), with application of an oscillating (F = 4 Hz) magnetic field. At 48 h post-transfection, cells were detached, counted (to assess cytotoxicity) and allowed to form neurospheres in culture medium with/without exogenous FGF2. (A) Bar chart of total cell count and (B) cell viability prior to neurosphere formation; (C) Bar chart showing neurosphere number and (D) size at 96 h after plating in neurosphere medium ± exogenous FGF2. *P < 0.05, **P < 0.01 and ***P < 0.001 (one-way ANOVA and Bonferroni’s MCT). (E) Bar chart showing total cell count after dissociation of neurospheres at 144 h after plating in neurosphere medium ± exogenous FGF2. *P < 0.05, **P < 0.01 and ***P < 0.001 (one-way ANOVA and Bonferroni’s MCT).
Mentions: In agreement with earlier findings, for all three plasmids, microscopy revealed no obvious adverse effects on cell morphology and cell adherence for cells treated with complexes compared with cells treated with plasmid alone. Further analysis of cells transfected under oscillating (F = 4 Hz) magnetic field conditions, i.e., conditions which yield the highest transfection efficiencies, revealed normal total counts and cell viability at 48 h after delivery of either pFGF2-GFP or pAN-GFP (Figure 5A,B), consistent with findings for pmaxGFP.

Bottom Line: 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).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