PONE-D-21-06129

Treatment of necrotizing enterocolitis by conditioned medium derived from human amniotic fluid stem cells

PLOS ONE

Dear Dr. Pierro

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.

In particular, the results of this study are of interest . However, the manuscript would increase merit if the potential mechanism of action of the CM in respect to cell therapy would be better detailed. Moreover, the identification of the CM dosage chosen needs rationale.

Please submit your revised manuscript by 25th July. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Benedetta Bussolati, MD, PhD

Academic Editor

PLOS ONE

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2. Thank you for stating the following in the Financial Disclosure section:

[AP is the recipient of a Canadian Institutes of Health Research (CIHR) Foundation Grant 353857. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.].    

We note that one or more of the authors are employed by a commercial company: Micregen Ltd.

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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: Partly

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: Joshua S. O’Connell et al. present a research on therapeutic effect of conditioned medium (CM) derived from human AFSC on NEC without risk of tumorgenesis as well as immunogenicity. The manuscript must describe a technically sound piece of scientific research with data that partly 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.It is quite interesting but remains some problems that needs to be addressed.

Detailed comments are as follows:

1. In line 61, what kinds of MSC does the author refer to? As postnatal MSC contain various types of MSC such as bone marrow-derived or adipose-derived mesenchymal stem cells.

2. Flow chart of the experiment is suggested.

3. Why does the author choose 100μL as the dose of CM to treat NEC? The author should explain.

4. Is there any difference of therapeutic efficacy between CM and MSC transplantation?

5. The author should add more experiments to examine the effect of CM on NEC such as apotosis or others.

6. As the effect of CM was so prominent, the author need to explain what composition in CM plays an key role in the CM-mediated therapeutic effect.

Reviewer #2: This manuscript by O’Connell JS et al. describes the protective effects of conditioned medium derived from human amniotic fluid stem cells (AFSC) in experimental mouse model of necrotizing enterocolitis (NEC). NEC was induced starting at post-natal day P5 via gavage feeding of hyperosmolar formular 4x a day, immediately following 10 minute hypoxia prior to all feeds and LPS administration on P6 and P7. For treatment, CM were administered on P6 and P7 at a dose of 100uL/each via IP injection; PBS administration were used as control. The experiment was terminated 2 days after the last administration of CM at post-natal day P9, and terminal ileum were harvested and processed for histologic evaluation and molecular characterization for IL-6, TNFalpha, Lgr5 gene expression. The authors concluded that the CM treatment group demonstrated less tissue damage, lower pro-inflammatory IL-6 and TNF-alpha expression and improved counts for stem cell activity marker Lgr5. The manuscript is well written and the research study address an important problem in neonatal gastroenterology. Knowledge gained from this study could potentially advance the field.

Major concerns:

1. The authors have previously published (ref. #14) that human AFSC derived extracellular vesicles provide benefits identical to the results reported herein Are the AFSC used in this study different from that used in reference #14, and if so, how? The authors refer to ref. #14 in relation to methods used and in the context of EVs in general as a potential mechanism of action in the CM, but the discussion falls short of clearly stating that those EVs were also derived from AFSC. It should be therefore clearly stated in the discussion how these two studies are different in terms of the stem cells used (if they are), otherwise it seems as if the hypothesis proposed herein has been previously tested. If the stem cells in both studies are of the same kind (not necessarily from the same source), then there is no novelty and/or significance to the present study, as in some respects it replicates previous work.

2. The data for gene expression for IL6, TNFa and Lgr5 are impressive, however, confirmation of this effect at protein level will be more convincing. VEGF is known to be decreased during NEC; have you assessed if treatment with CM helps restore VEGF levels? How about neutrophils and macrophages?

3. Also, adding an immunostaining for Lgr5 or rather a co-staining of Lgr5 and Ki67 together will be more valuable and informative to demonstrate intestinal repair and regeneration.

4. How was it determined that two doses of CM administration were needed to generate the beneficial effect? Is there evidence that single dose is not effective?

5. Does the CM treatment provide long term benefit, or the observed changes are transient? In this regard, a survival study will be very useful for demonstrating any lasting effects.

Minor concerns:

1. DAPI staining (described in methods) is missing in Figure 4.

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Reviewer #1: No

Reviewer #2: No

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Attachments

Attachment

Submitted filename: Review.docx

PONE-D-21-06129R1Treatment of necrotizing enterocolitis by conditioned medium derived from human amniotic fluid stem cellsPLOS ONE

Dear Dr.Pierro

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. In particular, although the manuscript is ameliorated, it still requires an improvement in the quality of data presentation and discussion,  typos to be corrected and figures to be referred in the text.

Please submit your revised manuscript by 12ve December. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Benedetta Bussolati, MD, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

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?

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

**********

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?

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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5. 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: No

**********

6. 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: (No Response)

Reviewer #2: This revised manuscript shows an improvement over the original submission, and the new data provided greatly enhances the interpretation of the results. However, the manuscript will further benefit by addressing the following:

1. Figure 2F-G are not referenced in the text.

2. Grammatical error in the following lines 230-232

3. Figure 2J is not referenced. The VEGF expression data is missing in the results. What is the conclusion from the VEGF data is not mentioned throughout the manuscript.

4. The DAPi staining in Figure 3C for the NEC +PBS group does not match with the rest of the stainings.

5. The sample size for the NEC + PBS group needs clarification. It is n=11 according to the dot plot in Figure 3E, but the legend states n=10. This needs to be clarified also in the methods, line 141-142.

6. Figure 5 illustration can be improved by providing the following:

a. description of the cells types shown

b. change of gene or pathway expression in the NEC +CM group similar to the NEC (or NEC + PBS)

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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.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Sargis Sedrakyan

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Treatment of necrotizing enterocolitis by conditioned medium derived from human amniotic fluid stem cells

PONE-D-21-06129R2

Dear Dr. Pierro,

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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Kind regards,

Benedetta Bussolati, MD, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: