• Summary of the article and findings:

Undifferentiated neural precursor cells (NPCs) have been proven to be electrosensitive, which means they have the ability to respond to electric fields. Previous studies have already demonstrated that direct current electric fields (dcEF) could lead to rapid and cathode-directed migration of NPCs, also known as galvanotaxis. This article investigated the usage of balanced biphasic bipolar pulses instead of dcEF, which has been suggested that it could promote cell survival and anti-apoptotic effects in growth factor-depleted conditions. It was proposed to be able to minimize the accumulation of charge and toxic by-products. In order to investigate the migratory properties of the NPCs cells with the application of biphasic stimulation, in vitro galvanotaxis chamber with NPCs extracted from mouse subventricular zone plated on a slide was stimulated. Live cell time-lapse imaging and cell-tracking techniques were used to analyze the migratory behaviour of the cells in the presence of the biphasic electric fields. In addition, similar methodology was used to compare the differences between biphasic bipolar and biphasic monopolar stimulation to induce galvanotaxis of NPCs. Undifferentiated adult mouse subependymal NPCs exposed to biphasic monopolar stimulation undergo rapid and directed migration toward the cathode. The results from the experiments suggested that balanced biphasic electric fields successfully induced galvanotaxis of on undifferentiated NPCs and the migration was directed towards the cathode. In addition, the cells only responded to biphasic monopolar stimulation and that differentiated NPCs fails to migrate with the application of electric fields.

• Overall strengths of the article and what impact it might have in your field:

The authors provided a clear and succinct background regarding what is already known in the field. Detailed information was provided for topics relating to the objective listed in the abstract and why it led to the research question of the article. The article was then able to answer its objective, supported by the data collected from the experiments performed. Figures were able to convey what the authors wanted to prove, mainly regarding the fact that the NPCs could migrate under the influence of biphasic stimulation. The results were discussed from various angles and were connected to what were suggested in other literature. By live-cell recording the cells’ migration in vitro, it was able to clearly trace the response to the electrical fields in real time. Also, the migration analysis software enabled the analysis of magnitude of velocity, directedness and tortuosity of migration. It allows better understanding of the response of the cells to the electric fields such as figuring out to what extend would the different parameters be altered.

Despite the effectiveness of dcEF in promoting galvanotaxis in undifferentiated NPCs, one of the major hurdles is that prolonged exposure to dcEF results in charge accumulation. This could cause electrode corrosion, the formation of toxic chemical species and tissue damage. The charged-balanced biphasic stimulation was suggested as an alternative that could minimize the adverse effects. In addition, the experiments demonstrated that only undifferentiated cells respond to the electrical stimulation. It provided insight for clinical applications as it limits the cell type that the stimulation could influence as well as leads to further investigations in terms of why the differentiated cell type fails to respond.

• Specific comments on weaknesses of the article and what could be done to improve it:

Major points:

1. In addition to the comparison between the biphasic monopolar, biphasic bipolar, and no stimulation, dcEF could also be included as one of the stimulation conditions. The same analysis of the cells in terms of their migratory behaviours could be performed for dcEF stimulation. This could allow clearer comparisons between the different stimulations and could act as a positive control as it has been proven that it does promote rapid NPC migration.

2. Since one of the major advantages of biphasic stimulation is that it can minimize the adverse effects from dcEF, the authors could better support their hypothesis if they could perform a cell survival analysis post-stimulation with dcEF or biphasic stimulation. This simple experiment could allow the authors to clearly demonstrate that biphasic stimulation can reduce toxicity via cell survival rate. This could be achieved with TUNEL assay.

3. As biphasic stimulation is the new proposed electrical stimulation, more description of how it works, the differences between dcEF and biphasic stimulation could be added as a part of the background in the introduction. There was only one citation regarding previous studies on biphasic stimulation, more articles on the topic could be provided for a more informative background and allow the audience to better understand the rationale behind selecting biphasic stimulation.

Minor points:

1. In the discussion, future direction was mentioned to be in vivo work. It is believed that there are other steps, such as assessing cell survival, proliferation, and differentiation in vitro, could also be included to verify the effects of biphasic stimulation before moving forward to in vivo work. The above could be achieved with immunocytochemistry staining with various proliferation markers as well as specific cell type markers.

2. Many other literatures used a 3 hours stimulation paradigm for NPCs instead of 6 hours and the reason for choosing this duration was not mentioned in the paper. It would be good to demonstrate cells’ migratory behaviour in 3 hours so that it is more comparable to other articles, aiding in making connections with other results to validate the authors' findings.

3. In Figure 3 and 4 A, B, C, the x-axis could also be labelled for better formatted diagrams, and allows the audience to have a better idea of what the x-axis is representing without reading the text.