PONE-D-19-14991

Investigating the potential of the secretome of mesenchymal stem cells derived from sickle cell disease patients

PLOS ONE

Dear Dr Fortuna,

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Carlos E. Ambrósio, Ph.D

Academic Editor

PLOS ONE

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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: 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: The Manuscript named “Investigating the potential of the secretome of mesenchymal stem cells derived from sickle cell disease patients” presented is well structured and designed.

I believe that the manuscript has potential to be publish after some clarifications and changes.

Abstract

Why did the authors affirmed that they tested a hypothetically molecules? Why did the authors test an unsure molecules?

Line 25: “in addition to the expression of paracrine molecules that hypothetically contribute to angiogenesis and skin regeneration”

Line 35: Please review this statement: “In sum, culturing under hypoxic conditions produced profound effects on the BMSC secretome, as demonstrated by the expression of trophic paracrine factors involved in angiogenesis and skin regeneration”.

Introduction

Line 59: “and promotes tissue healing and the formation of new blood vessels.”

Change and promotes for “, promote”. And the formation for “formation”

Line 60: the main bioactive factors ghange for “important bioactive factor”

Line 63: Please review this statement: “design highly potent proangiogenic MSC-based cell therapies”. The word design didn’t fit well with the maining.

Line 66- the gap: However, in SCD, 66 the key factors secreted by BMSCs that possess the potential to promote angiogenesis and tissue repair have not been identified to date.

Line 78: Please add the reference for- “The conditioned medium derived from the BMSC culture has been reported to serve multiple positive functions in tissue regeneration”.

Line 82: Please add the references for- “Although numerous studies using BMSCs and their conditioned mediums as potential therapeutic agents have been published”.

Material and Methods

Line 137: “The cells and the CM were then collected and processed at 3200 rpm for 20 min at 4 o C, and kept at -70 o C until use”.

Was 0.5% oxygen for 48h enough to really see a hypoxic result?

Could -70 C affect the results?

When the authors collected CM, was it filtered?

Did the authors remove extracellular vesicles (which are bioactive vesicles) from CM?

Line 150: “Relative mRNA expression of the target genes”

Was these mRNA isolated from CM or BMSC?

Line 190: After a 1-hour incubation period at 37 o C, the angioreactors were subcutaneously implanted into the dorsal flanks of 8-week-old 192 female C57/BL6 mice.

How many animals was used?

Line 281: In “were highly positive for surface” Please add the percentage of each marker.

How the authors did decide the secretome concertation/quantity for the in vivo study?

Results

The results sections are lined with the M & M.

Fig 3B add the STDEV or STD error on the graph.

Discussion

Line 460: In “Recent studies ….”, please add more references to this affirmative.

Other points:

-In general, the authors performed a great study with very interesting results. But it is unclear if they considered in the M & M the proteins contained in the extracellular vesicles as part of the analyzed proteins or not. I suggest to the authors to let clear this point in the M & M.

There as several studies indicating that extracellular vesicles have angiogenesis function:

Pro-Angiogenic Actions of CMC-Derived Extracellular Vesicles Rely on Selective Packaging of Angiopoietin 1 and 2, but Not FGF-2 and VEGF. Wysoczynski M, Pathan A, Moore JB 4th, Farid T, Kim J, Nasr M, Kang Y, Li H, Bolli R.Stem Cell Rev. 2019 May 17. doi: 10.1007/s12015-019-09891-6.

Extracellular Vesicles in Angiogenesis. Todorova D, Simoncini S, Lacroix R, Sabatier F, Dignat-George F. Circ Res. 2017 May 12;120(10):1658-1673. doi: 10.1161/CIRCRESAHA.117.309681.

-In the discussion the authors used the secretome as a general word to describe the compounds from CM, I believe that could be better to use the same concept in the material and methods.

-The authors could discuss better what is in secretome, and why to use a general cell secretion instead of only proteins and/or only extracellular vesicles?

Reviewer #2: In this study everything seems to be according the questions above. The manuscript is presented in standard and correct English, all the experiments appear to have been done appropriately and the conclusion is supported by the data presented.

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Reviewer #1: No

Reviewer #2: No

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Attachments

Attachment

Submitted filename: Minor revision requirements.pdf

Investigating the potential of the secretome of mesenchymal stem cells derived from sickle cell disease patients

PONE-D-19-14991R1

Dear Dr. Vitor Fortuna,

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

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Carlos E. Ambrósio, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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

**********

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: Yes

**********

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: The authors addressed all suggestions, and I believe that now the manuscript is in a good shape to be published.

Reviewer #2: In this study everything seems to be according the questions above.

The authors corrected all previous questions, the manuscript is presented in standard and correct English, all the experiments appear to have been done appropriately and the conclusion is supported by the data presented.

**********

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: No