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Specific intensity direct current (DC) electric field improves neural stem cell migration and enhances differentiation towards βIII-tubulin+ neurons

PLoS ONE (2015) 10(6)
DOI: 10.1371/journal.pone.0129625
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Abstract

Control of stem cell migration and differentiation is vital for efficient stem cell therapy. Literature reporting electric field-guided migration and differentiation is emerging. However, it is unknown if a field that causes cell migration is also capable of guiding cell differentiation - and the mechanisms for these processes remain unclear. Here, we report that a 115 V/m direct current (DC) electric field can induce directional migration of neural precursor cells (NPCs). Whole cell patching revealed that the cell membrane depolarized in the electric field, and buffering of extracellular calcium via EGTA prevented cell migration under these conditions. Immunocytochemical staining indicated that the same electric intensity could also be used to enhance differentiation and increase the percentage of cell differentiation into neurons, but not astrocytes and oligodendrocytes. The results indicate that DC electric field of this specific intensity is capable of promoting cell directional migration and orchestrating functional differentiation, suggestively mediated by calcium influx during DC field exposure.

Figures

  • Fig 1. Live cell imaging and grid measurement method to characterize cell migration kinematics.Using a 5mm x 5mm (converted to 15 μmX 15 μm actual size) imposed transparency grid, individual cell movement was analyzed in the electric field. Cells were directed to move towards the cathode (-) under 115 V/m DC electric field stimulation.
  • Fig 2. Whole-cell patch clamp recording of the cultured neural stem cell in 115 V/m DC electric field. (A) Experimental setup, including perfusion chamber, two Ag/AgCl electrodes, a 1.5 V battery, and a recording glass electrode. Uniformed electric field was generated by passing electric current between two parallel Ag/AgCl electrodes. Potential difference was generated by the 1.5V battery. (B) Electric potential was measured by moving the electrode from one Ag/AgCl wire to the other one with a step of 1 mm. (C) Plot of voltage change vs. the distance of electrode moved. The field intensity is 115 V/m. (D) For whole cell patching, cells were plated on the matrigel on a small coverslip (10 mm X 10 mm), which was positioned in the recording chamber. Signal was amplified by a 700B amplifier and recorded by a computer. (E) Whole cell patch electrode was applied to the cell under visual guidance.
  • Fig 3. Stemness and differentiation of the cultured NPCs. (A) Neurospheres formed after 9 days growth of the isolated NPCs from the neonatal mice in the presence of EGF, bFGF, and heparin in serum free medium. Cells expressed neuronal stem cell marker Nestin (red). DAPI (blue) was used for nuclei and chromosome counterstain. (B) Partially differentiated neurosphere showing Nestin+ (Red) and GFAP+ (Green) after 3 days growth in serum free medium in the presence of 1% Fetal Bovine Serum (FBS). (C) 9 days after differentiation, cells largely becameGFAP+ astrocytes (green), and a small amount of cells differentiated into βIII-tubulin+ neurons (red). Scale bar = 100 μm.
  • Fig 4. NPCs cultured in matrigel migrated in 115 V/m DC electric field (EF). (A) Kinematic analysis of cell migration in control, EF, and EF + EGTA groups. Distribution of cells at the end of the experiment in each experimental group. Plot here is the final relative location of cells assuming their original location is (x = 0; y = 0). (B) Percentage of mobile cells observed in each experimental group. (C) Distance of cell movement in horizontal (x) and vertical (y) directions after 90 minutes of electric field exposure. (D) Circular plots show the angle of cells’movement in control, EF and EF + EGTA groups. The radii of these plots represent the number of cells migrated in that specific angle. Cells demonstrated clear cathodal migration in the EF group.
  • Fig 5. Cell morphology change during EF-directed migration. EF field was implemented for 90 min in each trial. Yellow arrows indicate movement of the cell body, red arrows for hind processes, and blue arrows for forward processes based on direction of EF field. Negative sign (-) indicates location of the cathode and the desired direction of migration. (A) One sample cell. (A1) At time = 0, one hind process (red) and one forward process (blue) are found while the cell body (yellow) remains evenly distributed and centralized. (A2) At t = 45 min, the hind process begins to detach while a new forward process forms in the cathodal direction; the cell body begins to redistribute in the direction of the forward processes. (A3) At time = 90 min, the hind process is absorbed by the cell body, the two forward processes have grown in size and length, and the cell body has moved in the cathodal direction. (B) A second sample cell. (B1) At time = 25 min, three hind processes are attached to nearby NPCs, anchoring the cell; one forward process is attached and the cell body is centralized. (B2) At t = 40 min, one hind process is detached from NPC (red), cell body has shifted downward (yellow), and one additional forward process has formed in the direction of the cathode (blue). (B3) At t = 60 min, all three hind processes have detached, the two forward processes have increased in size and length, and cell body has shifted further downward in direction of cathode.
  • Fig 6. Intracellular administration of EGTA prevented the EF-mediated NPCmigration. (A) An example of successful dialysis of fluorescence indicator Lucifer Yellow into the NPC via a patch pipette. (B) The cell was injected with EGTA (1 mM) with the patching pipette with 5 minutes of delivery before it was withdrawn. Consequent application of 115 V/m DC electric field for 20 minutes didn’t cause noticeable cell shape change and movement.
  • Fig 7. Depolarization of the cultured NPCs by 115 V/m DC electric field. (A) The patched cell had a resting membrane potential of -59 mV. When the DC electric field was turned on, the patching electrode recorded a large transient voltage change, which then stabilized after 1 minute at -46 mV. (B) To estimate the artifacts recorded by the electrode, after patching experiment, the same electrode was used to record the voltage changes (7 mV) at an isopotential line, next to the cell. This stimulus artifact was removed from the intracellular recording to obtain the resting membrane potential of the cell inside the E-field (- 53mV). Therefore, the cell was depolarized by 6 mV in the E-field. (C) I-V relations recorded from the cell before, during, and after DC electric field exposure.
  • Fig 8. The undifferentiated NPCs did not express voltage-dependent calcium channels. (A) Voltage clamp recording. Cells were held at various potentials and the corresponding currents were recorded. (B) TTX (1.0 μM) was applied into the medium to block Na channels. (C) Both TTX (1.0 μM) and CdCl2 (250 μM) in the medium. No existing evidence demonstrates the inward current that corresponds to either Na + or CdCl2 sensitive channels (VDCCs). (D) Current-voltage relationship.

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Zhao, H., Steiger, A., Nohner, M., & Ye, H. (2015). Specific intensity direct current (DC) electric field improves neural stem cell migration and enhances differentiation towards βIII-tubulin+ neurons. PLoS ONE, 10(6). https://doi.org/10.1371/journal.pone.0129625

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