Limits...
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

Balanced biphasic stimulation does not elicit galvanotaxis in neural precursor cells induced to differentiate. (A, B, C) Differentiated cells exhibit similar magnitude of velocity (/velocity/) (A), directedness (B) and tortuosity (C) of migration when exposed to biphasic monopolar (BPMP) stimulation (n = 3), biphasic bipolar (BPBP) stimulation (n = 3) or no stimulation (n = 3). (D, E, F) Individual cell migration tracks localized to a common origin show that differentiated cells undergo little migration over 6 hours of time-lapse imaging in the presence of BPMP stimulation (D), BPBP stimulation (E) or in the absence of stimulation (F). (G, H, I, J, K, L) Differentiated cells express β-III tubulin (G, H; arrowheads) or glial fibrillary acidic protein (GFAP)(I, J) prior to (G, I) and following (H, J) 6 hours of time-lapse imaging, when exposed to biphasic electrical stimulation, or not stimulated (K, L). (G′, H′, J′, K′, L′) Higher magnification images of the regions within the dashed boxes in (G to L). Scale bars = 200 μm. Data presented as mean ± standard error of the mean. *P <0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Balanced biphasic stimulation does not elicit galvanotaxis in neural precursor cells induced to differentiate. (A, B, C) Differentiated cells exhibit similar magnitude of velocity (/velocity/) (A), directedness (B) and tortuosity (C) of migration when exposed to biphasic monopolar (BPMP) stimulation (n = 3), biphasic bipolar (BPBP) stimulation (n = 3) or no stimulation (n = 3). (D, E, F) Individual cell migration tracks localized to a common origin show that differentiated cells undergo little migration over 6 hours of time-lapse imaging in the presence of BPMP stimulation (D), BPBP stimulation (E) or in the absence of stimulation (F). (G, H, I, J, K, L) Differentiated cells express β-III tubulin (G, H; arrowheads) or glial fibrillary acidic protein (GFAP)(I, J) prior to (G, I) and following (H, J) 6 hours of time-lapse imaging, when exposed to biphasic electrical stimulation, or not stimulated (K, L). (G′, H′, J′, K′, L′) Higher magnification images of the regions within the dashed boxes in (G to L). Scale bars = 200 μm. Data presented as mean ± standard error of the mean. *P <0.05.

Mentions: We previously demonstrated that when NPCs are induced to differentiate into neural phenotypes, they lose their ability to undergo galvanotactic migration in the presence of DCEFs [26]. We next asked whether differentiated cells would undergo galvanotaxis when exposed to BPMP or BPBP pulses. Neurospheres (passages 0 to 4) were plated onto Matrigel-coated galvanotaxis chambers in the presence of 1% FBS for 72 to 96 hours to induce cell differentiation. Differentiated cells were maintained in 1% FBS conditions and time-lapse imaged for 2.5 to 6 hours while either stimulated with BPMP or BPBP pulses, or not stimulated. Differentiated cells did not undergo galvanotaxis either in the presence or the absence of biphasic stimulation (both BPMP and BPBP modes), consistent with our previous observations with DCEFs. The /velocity/ (BPBP: 0.07 ± 0.01 μm/minute, BPMP: 0.06 ± 0.01 μm/minute), directedness (BPBP: 0.07 ± 0.17, BPMP: 0.09 ± 0.07) and tortuosity (BPBP: 4.53 ± 0.58, BPMP: 2.85 ± 0.68) of migration did not significantly differ from that of nonstimulated differentiated cells (/velocity/: 0.06 ± 0.01 μm/minute, directedness: −0.04 ± 0.04, tortuosity: 2.39 ± 1.01) (Figure 4A,B,C,D,E,F; Additional file 6). Similar to undifferentiated cells, this analysis was repeated throughout the entire time lapse in 30-minute increments (Additional file 7). Immunocytochemical analysis verified that the cells had differentiated into mature phenotypes prior to time-lapse imaging, and their mature phenotypes were maintained after 6 hours of imaging in the presence or absence of biphasic stimulation (Figure 4G,H,I,J,K,L). We conclude that biphasic stimulation does not induce galvanotaxis in differentiated neural cells.Figure 4


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)

Balanced biphasic stimulation does not elicit galvanotaxis in neural precursor cells induced to differentiate. (A, B, C) Differentiated cells exhibit similar magnitude of velocity (/velocity/) (A), directedness (B) and tortuosity (C) of migration when exposed to biphasic monopolar (BPMP) stimulation (n = 3), biphasic bipolar (BPBP) stimulation (n = 3) or no stimulation (n = 3). (D, E, F) Individual cell migration tracks localized to a common origin show that differentiated cells undergo little migration over 6 hours of time-lapse imaging in the presence of BPMP stimulation (D), BPBP stimulation (E) or in the absence of stimulation (F). (G, H, I, J, K, L) Differentiated cells express β-III tubulin (G, H; arrowheads) or glial fibrillary acidic protein (GFAP)(I, J) prior to (G, I) and following (H, J) 6 hours of time-lapse imaging, when exposed to biphasic electrical stimulation, or not stimulated (K, L). (G′, H′, J′, K′, L′) Higher magnification images of the regions within the dashed boxes in (G to L). Scale bars = 200 μm. Data presented as mean ± standard error of the mean. *P <0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Balanced biphasic stimulation does not elicit galvanotaxis in neural precursor cells induced to differentiate. (A, B, C) Differentiated cells exhibit similar magnitude of velocity (/velocity/) (A), directedness (B) and tortuosity (C) of migration when exposed to biphasic monopolar (BPMP) stimulation (n = 3), biphasic bipolar (BPBP) stimulation (n = 3) or no stimulation (n = 3). (D, E, F) Individual cell migration tracks localized to a common origin show that differentiated cells undergo little migration over 6 hours of time-lapse imaging in the presence of BPMP stimulation (D), BPBP stimulation (E) or in the absence of stimulation (F). (G, H, I, J, K, L) Differentiated cells express β-III tubulin (G, H; arrowheads) or glial fibrillary acidic protein (GFAP)(I, J) prior to (G, I) and following (H, J) 6 hours of time-lapse imaging, when exposed to biphasic electrical stimulation, or not stimulated (K, L). (G′, H′, J′, K′, L′) Higher magnification images of the regions within the dashed boxes in (G to L). Scale bars = 200 μm. Data presented as mean ± standard error of the mean. *P <0.05.
Mentions: We previously demonstrated that when NPCs are induced to differentiate into neural phenotypes, they lose their ability to undergo galvanotactic migration in the presence of DCEFs [26]. We next asked whether differentiated cells would undergo galvanotaxis when exposed to BPMP or BPBP pulses. Neurospheres (passages 0 to 4) were plated onto Matrigel-coated galvanotaxis chambers in the presence of 1% FBS for 72 to 96 hours to induce cell differentiation. Differentiated cells were maintained in 1% FBS conditions and time-lapse imaged for 2.5 to 6 hours while either stimulated with BPMP or BPBP pulses, or not stimulated. Differentiated cells did not undergo galvanotaxis either in the presence or the absence of biphasic stimulation (both BPMP and BPBP modes), consistent with our previous observations with DCEFs. The /velocity/ (BPBP: 0.07 ± 0.01 μm/minute, BPMP: 0.06 ± 0.01 μm/minute), directedness (BPBP: 0.07 ± 0.17, BPMP: 0.09 ± 0.07) and tortuosity (BPBP: 4.53 ± 0.58, BPMP: 2.85 ± 0.68) of migration did not significantly differ from that of nonstimulated differentiated cells (/velocity/: 0.06 ± 0.01 μm/minute, directedness: −0.04 ± 0.04, tortuosity: 2.39 ± 1.01) (Figure 4A,B,C,D,E,F; Additional file 6). Similar to undifferentiated cells, this analysis was repeated throughout the entire time lapse in 30-minute increments (Additional file 7). Immunocytochemical analysis verified that the cells had differentiated into mature phenotypes prior to time-lapse imaging, and their mature phenotypes were maintained after 6 hours of imaging in the presence or absence of biphasic stimulation (Figure 4G,H,I,J,K,L). We conclude that biphasic stimulation does not induce galvanotaxis in differentiated neural cells.Figure 4

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