Hypoxic Preconditioned Neural Stem Cell-Derived Extracellular Vesicles Contain Distinct Protein Cargo from Their Normal Counterparts
Round 1
Reviewer 1 Report
In the current manuscript the authors examined the role of hypoxia pre-conditioning on survival of neural stem cells through secretion of extracellular vesicle. However the authors need to address the following concerns to establish their findings.
1. Hypoxia pre-conditioning induced cell viability is very small. there is only 0.1 to 0.3 fold increase which is not convincing enough.
2. Authors have provided no evidence for the role of EV in induced cell viability. The observed increase in cell viability may be due to other growth factors secreted during hypoxia (like VEGF).
3. To establish the fact that Hypoxia pre-conditioning induced EV secretion is beneficial, authors need to treat stressed neuronal cells with isolated EVs and check cell survival.
4. Sox2 staining in neuro-spheres is not good.
Author Response
Dear reviewer, thank you very much for revising our paper. We highly appreciate your precious suggestions and we responded to them one by one as below:
- Hypoxia pre-conditioning induced cell viability is very small. there is only 0.1 to 0.3 fold increase which is not convincing enough.
Respond: Dear reviewer, thank you very much for your comment. Since we only wanted to trigger the cells with a mediate amount of oxygen we did not seek a huge change. We wished to show that small changes in oxygen can also make changes in the genetic contents of the EVs.
- Authors have provided no evidence for the role of EV in induced cell viability. The observed increase in cell viability may be due to other growth factors secreted during hypoxia (like VEGF).
Respond: Dear reviewer, we agree that confirming the increase of cell viability caused by EV would make it a more complete study and it is our future plan. However, the purpose of this study was to observe the changes in EVs after hypoxic preconditioning. We will investigate the effect of EVs on cell viability in our future study.
- To establish the fact that Hypoxia pre-conditioning induced EV secretion is beneficial, authors need to treat stressed neuronal cells with isolated EVs and check cell survival.
Respond: Dear reviewer, we agree that confirming that the hypoxic preconditioning-induced EV section is beneficial for neurons is a great idea and it is our future plan to carry it out. However, the purpose of the current study was to observe the genetic content of EVs changes after hypoxic preconditioning. We will investigate the effect of EVs on neuron viability in our future study.
- Sox2 staining in neuro-spheres is not good.
Respond: Dear reviewer, thank you very much for pointing this out. We appreciate your comment and we added new SOX2 staining for neurospheres.
Reviewer 2 Report
Review for “Hypoxic preconditioned neural stem cell derived extracellular vesicles contain distinct protein cargo from their normal counterparts”
The authors show the presence of several neuronal survival proteins in the EVs isolated after hypoxic preconditioning. It is an interesting study describing the protein composition of mouse NSC derived EVs. Here below are some minor issues to address:
1. Line 63, characterization was done based on markers for different cell sub types but not on functional properties. Would be good to rephrase “functional characteristics”.
2. Line 71: “Immunostaining identify the primary mouse cortex NSCs.” It is not clear?
3. Scale bars in Figure 3F are not visible
4. Would be nice to describe the method part of how proteomic data analysis was performed?
5. It is not clear what is n in figure legends, please specify it.
6. Is it n = 4 or n =6 (text vs figure 4A legend) used for the proteomic analysis?
7. Line 350-351, it is confusing with the method of cDNA synthesis, where two different cDNA kits are mentioned, please write it clearly.
8. Supplemental figure, panel labelling is missing.
9. Discussion is only focused on upregulated proteins, please also discuss the downregulated ones.
Author Response
Dear reviewer, thank you very much for revising our paper. We highly appreciate your precious suggestions and we responded to them one by one as below:
- Line 63, characterization was done based on markers for different cell sub types but not on functional properties. Would be good to rephrase “functional characteristics”.
Respond: Dear reviewer, thank you very much for your comment. The sentence has been edited.
- Line 71: “Immunostaining identify the primary mouse cortex NSCs.” It is not clear?
Respond: Dear reviewer, thank you very much for pointing it out. The “identify” was changed to “identification”.
- Scale bars in Figure 3F are not visible
Respond: Dear reviewer, thank you very much for pointing it out. The scale bars have been made bigger and the number is written in the figure description.
- Would be nice to describe the method part of how proteomic data analysis was performed?
Respond: Dear reviewer, thank you very much for your suggestions. Data analysis of proteomics has been added.
- It is not clear what is n in figure legends, please specify it.
Respond: Dear reviewer, thank you very much for your suggestions. N in figure legends and text have been described.
- Is it n = 4 or n =6 (text vs figure 4A legend) used for the proteomic analysis?
Respond: Dear reviewer, thank you very much for pointing it out. N=6, and it has been edited in the text.
- Line 350-351, it is confusing with the method of cDNA synthesis, where two different cDNA kits are mentioned, please write it clearly.
Respond: Dear reviewer, thank you very much for pointing it out and sorry for the misunderstanding. The text has been edited.
- Supplemental figure, panel labelling is missing.
Respond: Dear reviewer, thank you very much for your comment. The panel labeling has been added to supplemental figures.
- Discussion is only focused on upregulated proteins, please also discuss the downregulated ones.
Respond: Dear reviewer, thank you very much for pointing it out. The discussion concerning down-regulated proteins has been added.
