Limits...
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.


Effects of transfection protocols on cell viability and neurosphere formation. Monolayers (n = 4 cultures) were transfected with Neuromag-pmaxGFP complexes or with pmaxGFP only for controls, with application of the indicated magnetic fields. After 48 h, cells were detached from wells and a small proportion stained with trypan blue. (A) Bar chart showing the total number of cells per well. (B) Bar chart showing the proportion of viable cells. (C) Representative phase-contrast image of neurospheres formed from monolayers treated with particle/plasmid complexes; inset shows neurospheres formed from monolayers treated with plasmid only. (D) Fluorescence micrograph of neurospheres shown in (C), demonstrating GFP expression at 9 days post-transfection. (E) Bar chart showing the average sphere number per microscopic field. (F) Bar chart showing the average sphere diameter. Scale bar = 100 µm in (C,D).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4493511&req=5

jfb-06-00259-f002: Effects of transfection protocols on cell viability and neurosphere formation. Monolayers (n = 4 cultures) were transfected with Neuromag-pmaxGFP complexes or with pmaxGFP only for controls, with application of the indicated magnetic fields. After 48 h, cells were detached from wells and a small proportion stained with trypan blue. (A) Bar chart showing the total number of cells per well. (B) Bar chart showing the proportion of viable cells. (C) Representative phase-contrast image of neurospheres formed from monolayers treated with particle/plasmid complexes; inset shows neurospheres formed from monolayers treated with plasmid only. (D) Fluorescence micrograph of neurospheres shown in (C), demonstrating GFP expression at 9 days post-transfection. (E) Bar chart showing the average sphere number per microscopic field. (F) Bar chart showing the average sphere diameter. Scale bar = 100 µm in (C,D).

Mentions: Assessment of the safety of the developed protocols was limited to three magnetic field conditions: (i) no field, yielding the lowest transfection efficiency; (ii) static field (F = 0 Hz), currently the most widely used method for magnetofection; and (iii) oscillating field of F = 4 Hz, yielding the highest transfection efficiency. Compared with plasmid only controls, Neuromag-pmaxGFP complex addition had no effect on total cell number (Figure 2A) or viability (Figure 2B) at 48 h, irrespective of the magnetic field condition. For more stringent examination of toxicity, a neurosphere formation assay was employed in which the ability of transfected cells to form neurospheres was tested at 48 h post-transfection, with neurospheres allowed to form for 7 days. This biological assay allows for functional evaluation of cell stemness/proliferative capacity, which is key to the regenerative capacity of a transplant population such as the NSCs. Cells from treated cultures formed neurospheres (Figure 2C), which appeared morphologically similar to those from control cultures (Figure 2C, inset) with extensive GFP expression in neurospheres from transfected cultures (Figure 2D). Neurosphere number (Figure 2E) and size (Figure 2F) were similar between control and treated samples under all magnetic field conditions, indicating that the transfection protocols had no adverse effects on NSC self-renewal. In all experiments, the applied magnetic fields per se had no effect on cell number or viability (Figure 2A,B) or neurosphere formation (Figure 2E,F).


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)

Effects of transfection protocols on cell viability and neurosphere formation. Monolayers (n = 4 cultures) were transfected with Neuromag-pmaxGFP complexes or with pmaxGFP only for controls, with application of the indicated magnetic fields. After 48 h, cells were detached from wells and a small proportion stained with trypan blue. (A) Bar chart showing the total number of cells per well. (B) Bar chart showing the proportion of viable cells. (C) Representative phase-contrast image of neurospheres formed from monolayers treated with particle/plasmid complexes; inset shows neurospheres formed from monolayers treated with plasmid only. (D) Fluorescence micrograph of neurospheres shown in (C), demonstrating GFP expression at 9 days post-transfection. (E) Bar chart showing the average sphere number per microscopic field. (F) Bar chart showing the average sphere diameter. Scale bar = 100 µm in (C,D).
© Copyright Policy
Related In: Results  -  Collection

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

jfb-06-00259-f002: Effects of transfection protocols on cell viability and neurosphere formation. Monolayers (n = 4 cultures) were transfected with Neuromag-pmaxGFP complexes or with pmaxGFP only for controls, with application of the indicated magnetic fields. After 48 h, cells were detached from wells and a small proportion stained with trypan blue. (A) Bar chart showing the total number of cells per well. (B) Bar chart showing the proportion of viable cells. (C) Representative phase-contrast image of neurospheres formed from monolayers treated with particle/plasmid complexes; inset shows neurospheres formed from monolayers treated with plasmid only. (D) Fluorescence micrograph of neurospheres shown in (C), demonstrating GFP expression at 9 days post-transfection. (E) Bar chart showing the average sphere number per microscopic field. (F) Bar chart showing the average sphere diameter. Scale bar = 100 µm in (C,D).
Mentions: Assessment of the safety of the developed protocols was limited to three magnetic field conditions: (i) no field, yielding the lowest transfection efficiency; (ii) static field (F = 0 Hz), currently the most widely used method for magnetofection; and (iii) oscillating field of F = 4 Hz, yielding the highest transfection efficiency. Compared with plasmid only controls, Neuromag-pmaxGFP complex addition had no effect on total cell number (Figure 2A) or viability (Figure 2B) at 48 h, irrespective of the magnetic field condition. For more stringent examination of toxicity, a neurosphere formation assay was employed in which the ability of transfected cells to form neurospheres was tested at 48 h post-transfection, with neurospheres allowed to form for 7 days. This biological assay allows for functional evaluation of cell stemness/proliferative capacity, which is key to the regenerative capacity of a transplant population such as the NSCs. Cells from treated cultures formed neurospheres (Figure 2C), which appeared morphologically similar to those from control cultures (Figure 2C, inset) with extensive GFP expression in neurospheres from transfected cultures (Figure 2D). Neurosphere number (Figure 2E) and size (Figure 2F) were similar between control and treated samples under all magnetic field conditions, indicating that the transfection protocols had no adverse effects on NSC self-renewal. In all experiments, the applied magnetic fields per se had no effect on cell number or viability (Figure 2A,B) or neurosphere formation (Figure 2E,F).

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.