PONE-D-24-38324Extracellular vesicles from adipose-derived mesenchymal stem cells promote colony formation ability and EMT of corneal limbal epithelial cellsPLOS ONE

Dear Dr. Hayashi,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

ACADEMIC EDITOR: 

This paper makes a significant contribution to the field of corneal regeneration and stem cell therapy by exploring the use of AdMSC-EVs and their miRNAs. While there are areas that need further clarification and discussion, the overall experimental design is solid, and the findings provide a promising foundation for future studies in regenerative medicine. With additional in vivo validation and deeper mechanistic insights, this study could have a substantial impact on the development of EV-based therapies for corneal diseases.

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Mohamed A. M. Ali, Ph.D.

Academic Editor

PLOS ONE

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Additional Editor Comments:

� Expand Discussion on Mechanisms : The role of miRNAs in mediating the observed effects is central to the study, but further elucidation of the exact signaling pathways impacted by miR-25, miR-191, and miR-335 would be valuable. Are these miRNAs directly involved in the regulation of known EMT or stemness-related pathways?

� In Vivo Studies : Consider including in vivo models in future work to validate the therapeutic potential of AdMSC-EVs in a clinical setting. This will enhance the translational relevance of the findings.

� Clarify the Relationship between Proliferation and Colony Formation : More explanation is needed to reconcile the seemingly paradoxical observation that EVs reduce proliferation while enhancing colony formation. Are there alternative explanations, such as altered cell migration or differentiation status?

� Link EMT with Stemness : If EMT is driving regenerative properties, the manuscript should make a stronger case for the connection between these two processes. Consider referencing more literature that links EMT with stem cell maintenance, especially in epithelial tissues.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In methods the authors continuously referred “as previously described” but no reference is added.

Figure 1, EV 30ug/mL shows statistically significant data than the EV 120 ugl/mL. Authors need to clarify this, or need to have more control over experimental data, especially SD.

Figure 2, the data provided don’t support each other. Colony formation is increasing but proliferation is decreased. Colony formation and proliferation are directly related to each other, if colony is increasing its direct indication of proliferation.

Figure 4, the authors transfected the cell with miR individually, there must be a group where all the miR in cocktail are transfected and then investigate the desired effect.

Overall the manuscript show quality data, and nicely drawn conclusion. Authors are required to addressed the highlighted issues.

Reviewer #2: Authors have shown the effect of AdMSCs Evs on LEcs, the manuscript has shown interesting findings. But there are a few concerns with the manuscript that have to be addressed and revised.

1. Authors are suggested to mention the functional aspect of EVs diversely by briefly mentioning their role in cancer, immunology, etc. Authors are advised to cite these publications. PMID: 36713536, PMID: 36570199.

2. Authors are suggested to mention the results only in the result section and the figure legend should be included in the last.

3. The images of electron microscopy 1B, is of low quality.

The images of Figure 2 E are also suggested to be replaced by high-quality images.

4. Figure 4D, images are to be replaced with high quality, nothing is visible in the pdf.

5. The discussion should be shortened.

6. Supplementary figures legend should be added in supplementary files only.

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Reviewer #1: No

Reviewer #2: Yes:  Namrata Anand

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Extracellular vesicles from adipose-derived mesenchymal stem cells promote colony formation ability and EMT of corneal limbal epithelial cells

PONE-D-24-38324R1

Dear Dr. Hayashi,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Mohamed A. M. Ali, Ph.D.

Academic Editor

PLOS ONE

Reviewers' comments:

1. Have the authors have adequately addressed your comments raised in a previous round of review?

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Reviewer #1: The authors addressed the question raised during the review process; therefore, the manuscript is accepted in its current form.

Reviewer #2: The authors addressed the question raised during the review process; therefore, the manuscript is accepted in its current form.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no,” your identity will remain anonymous, but your review may still be made public.

Reviewer #1: No

Reviewer #2: Yes

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