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Biphasic monopolar electrical stimulation induces rapid and directed galvanotaxis in adult subependymal neural precursors.

Babona-Pilipos R, Pritchard-Oh A, Popovic MR, Morshead CM - Stem Cell Res Ther (2015)

Bottom Line: Neurospheres were plated onto galvanotaxis chambers in conditions that either promoted maintenance in an undifferentiated state or promoted differentiation into mature phenotypes.Single cell migration was subsequently tracked and the cells' magnitude of velocity, directedness and tortuosity were quantified.We demonstrate, for the first time, the use of balanced biphasic electric fields to induce galvanotaxis of NPCs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College Street, Room 407, M5S 3G9, Toronto, Ontario, Canada. robert.babona.pilipos@mail.utoronto.ca.

ABSTRACT

Introduction: Following injury such as stroke, adult mammalian subependymal neural precursor cells (NPCs) are induced to proliferate and migrate toward the lesion site where they differentiate into neural cells, albeit with limited efficacy. We are interested in enhancing this migratory ability of NPCs with the long-term goal of promoting neural repair. Herein we build on our previous studies demonstrating that direct current electric fields (DCEFs) promote rapid and cathode-directed migration of undifferentiated adult NPCs (but not differentiated phenotypes) - a phenomenon known as galvanotaxis. While galvanotaxis represents a promising strategy to promote NPC recruitment to lesion sites, stimulation of neural tissue with DCEFs is not a clinically-viable strategy due to the associated accumulation of charge and toxic byproducts. Balanced biphasic waveforms prevent the accumulation of charge and thus are outside of the limitations of DCEFs. In this study, we investigated the effects of balanced biphasic electrical stimulation on the migratory behaviour of undifferentiated subependymal NPCs and their differentiated progeny.

Methods: NPCs were isolated from the subependymal zone of adult mouse brains and cultured in a NPC colony-forming assay to form neurospheres. Neurospheres were plated onto galvanotaxis chambers in conditions that either promoted maintenance in an undifferentiated state or promoted differentiation into mature phenotypes. Chambers containing cells were then time-lapse imaged in the presence of either biphasic monopolar, or biphasic bipolar electrical stimulation, or in the complete absence of electrical stimulation. Single cell migration was subsequently tracked and the cells' magnitude of velocity, directedness and tortuosity were quantified.

Results: We demonstrate, for the first time, the use of balanced biphasic electric fields to induce galvanotaxis of NPCs. Undifferentiated adult mouse subependymal NPCs exposed to biphasic monopolar stimulation undergo rapid and directed migration toward the cathode. In contrast, both biphasic bipolar stimulation and the lack of electrical stimulation produced non-directed migration of NPCs. Notably, NPCs induced to differentiate into mature phenotypes prior to exposure to electrical stimulation do not migrate in the presence or absence of biphasic stimulation.

Conclusion: We purport that balanced biphasic stimulation represents a clinically-viable technique for mobilizing NPCs that may be integrated into strategies for promoting endogenous neurorepair.

No MeSH data available.


Related in: MedlinePlus

Illustration of experimental setup for galvanotaxis assay. Neural precursor cells plated in a galvanotaxis chamber are placed in the center of the stage of a live-cell imaging system. On either side of the chamber is a Petri dish containing serum-free media (SFM) and housing the Ag/AgCl electrode. The adjacent Petri dishes and the galvanotaxis chamber are bridged via agarose gel electrodes. The Compex stimulator is connected to the Ag/AgCl electrodes to provide biphasic monopolar or biphasic bipolar stimulation. Modified with permission from Babona-Pilipos and colleagues [25]. EFH, epidermal growth factor, basic fibroblast growth factor and heparin; FBS, fetal bovine serum.
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Fig2: Illustration of experimental setup for galvanotaxis assay. Neural precursor cells plated in a galvanotaxis chamber are placed in the center of the stage of a live-cell imaging system. On either side of the chamber is a Petri dish containing serum-free media (SFM) and housing the Ag/AgCl electrode. The adjacent Petri dishes and the galvanotaxis chamber are bridged via agarose gel electrodes. The Compex stimulator is connected to the Ag/AgCl electrodes to provide biphasic monopolar or biphasic bipolar stimulation. Modified with permission from Babona-Pilipos and colleagues [25]. EFH, epidermal growth factor, basic fibroblast growth factor and heparin; FBS, fetal bovine serum.

Mentions: We asked whether BPMP pulses could elicit a galvanotactic response in undifferentiated NPCs. We plated neurospheres (passages 0 to 4) onto galvanotaxis chambers for 17 to 20 hours in the presence of growth factors (SFM + EFH) to maintain the NPCs in an undifferentiated state. During this period, neurospheres adhered to the Matrigel-coated base of the chambers and individual NPCs moved radially away from the plated neurosphere. The migration of undifferentiated NPCs was then analyzed using time-lapse imaging for a period of 2.5 to 6 hours in the presence of BPMP or BPBP stimulation, as well as without any applied electric field. Figure 2 illustrates the experimental setup of the apparatus.Figure 2


Biphasic monopolar electrical stimulation induces rapid and directed galvanotaxis in adult subependymal neural precursors.

Babona-Pilipos R, Pritchard-Oh A, Popovic MR, Morshead CM - Stem Cell Res Ther (2015)

Illustration of experimental setup for galvanotaxis assay. Neural precursor cells plated in a galvanotaxis chamber are placed in the center of the stage of a live-cell imaging system. On either side of the chamber is a Petri dish containing serum-free media (SFM) and housing the Ag/AgCl electrode. The adjacent Petri dishes and the galvanotaxis chamber are bridged via agarose gel electrodes. The Compex stimulator is connected to the Ag/AgCl electrodes to provide biphasic monopolar or biphasic bipolar stimulation. Modified with permission from Babona-Pilipos and colleagues [25]. EFH, epidermal growth factor, basic fibroblast growth factor and heparin; FBS, fetal bovine serum.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4413998&req=5

Fig2: Illustration of experimental setup for galvanotaxis assay. Neural precursor cells plated in a galvanotaxis chamber are placed in the center of the stage of a live-cell imaging system. On either side of the chamber is a Petri dish containing serum-free media (SFM) and housing the Ag/AgCl electrode. The adjacent Petri dishes and the galvanotaxis chamber are bridged via agarose gel electrodes. The Compex stimulator is connected to the Ag/AgCl electrodes to provide biphasic monopolar or biphasic bipolar stimulation. Modified with permission from Babona-Pilipos and colleagues [25]. EFH, epidermal growth factor, basic fibroblast growth factor and heparin; FBS, fetal bovine serum.
Mentions: We asked whether BPMP pulses could elicit a galvanotactic response in undifferentiated NPCs. We plated neurospheres (passages 0 to 4) onto galvanotaxis chambers for 17 to 20 hours in the presence of growth factors (SFM + EFH) to maintain the NPCs in an undifferentiated state. During this period, neurospheres adhered to the Matrigel-coated base of the chambers and individual NPCs moved radially away from the plated neurosphere. The migration of undifferentiated NPCs was then analyzed using time-lapse imaging for a period of 2.5 to 6 hours in the presence of BPMP or BPBP stimulation, as well as without any applied electric field. Figure 2 illustrates the experimental setup of the apparatus.Figure 2

Bottom Line: Neurospheres were plated onto galvanotaxis chambers in conditions that either promoted maintenance in an undifferentiated state or promoted differentiation into mature phenotypes.Single cell migration was subsequently tracked and the cells' magnitude of velocity, directedness and tortuosity were quantified.We demonstrate, for the first time, the use of balanced biphasic electric fields to induce galvanotaxis of NPCs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College Street, Room 407, M5S 3G9, Toronto, Ontario, Canada. robert.babona.pilipos@mail.utoronto.ca.

ABSTRACT

Introduction: Following injury such as stroke, adult mammalian subependymal neural precursor cells (NPCs) are induced to proliferate and migrate toward the lesion site where they differentiate into neural cells, albeit with limited efficacy. We are interested in enhancing this migratory ability of NPCs with the long-term goal of promoting neural repair. Herein we build on our previous studies demonstrating that direct current electric fields (DCEFs) promote rapid and cathode-directed migration of undifferentiated adult NPCs (but not differentiated phenotypes) - a phenomenon known as galvanotaxis. While galvanotaxis represents a promising strategy to promote NPC recruitment to lesion sites, stimulation of neural tissue with DCEFs is not a clinically-viable strategy due to the associated accumulation of charge and toxic byproducts. Balanced biphasic waveforms prevent the accumulation of charge and thus are outside of the limitations of DCEFs. In this study, we investigated the effects of balanced biphasic electrical stimulation on the migratory behaviour of undifferentiated subependymal NPCs and their differentiated progeny.

Methods: NPCs were isolated from the subependymal zone of adult mouse brains and cultured in a NPC colony-forming assay to form neurospheres. Neurospheres were plated onto galvanotaxis chambers in conditions that either promoted maintenance in an undifferentiated state or promoted differentiation into mature phenotypes. Chambers containing cells were then time-lapse imaged in the presence of either biphasic monopolar, or biphasic bipolar electrical stimulation, or in the complete absence of electrical stimulation. Single cell migration was subsequently tracked and the cells' magnitude of velocity, directedness and tortuosity were quantified.

Results: We demonstrate, for the first time, the use of balanced biphasic electric fields to induce galvanotaxis of NPCs. Undifferentiated adult mouse subependymal NPCs exposed to biphasic monopolar stimulation undergo rapid and directed migration toward the cathode. In contrast, both biphasic bipolar stimulation and the lack of electrical stimulation produced non-directed migration of NPCs. Notably, NPCs induced to differentiate into mature phenotypes prior to exposure to electrical stimulation do not migrate in the presence or absence of biphasic stimulation.

Conclusion: We purport that balanced biphasic stimulation represents a clinically-viable technique for mobilizing NPCs that may be integrated into strategies for promoting endogenous neurorepair.

No MeSH data available.


Related in: MedlinePlus