PONE-D-23-39123Hair-follicle associated pluripotent (HAP)-cell-sheet implantation enhanced wound healing in diabetic db/db micePLOS ONE  Dear Dr. Hasegawa-Haruki, 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. This research paper, titled "Hair Follicle-Associated Pluripotent (HAP) Stem Cell Sheet Transplantation Enhances Wound Healing in Diabetic db/db Mice," explores the treatment of chronic ulcers, particularly diabetic wounds, using Hair Follicle-Associated Pluripotent (HAP) stem cells. The findings indicate that autologous HAP stem cell sheets have potential clinical value in treating refractory diabetic ulcers. I'm including feedback from two expert reviewers regarding your manuscript. Their insights highlight the study's potential while also presenting various critiques and recommendations. After read reviewer’s feedback, I believe this article requires careful revision. This article falls short in several aspects, including whether it is a significant contribution to the field, its organization and comprehensive description, its scientific soundness and accuracy, and the correctness and readability of the English used. All of these areas require a round of revisions for improvement. So, I decide give you major revision decision. It's our hope that you find this feedback constructive. Should additional data be available to address the raised concerns, we are open to examining a modified submission. Our timeline is adaptable for any necessary extended research. For submissions linked to a specific thematic issue, please liaise with the editorial team concerning resubmission deadlines. We emphasize that key findings must be backed by comprehensive statistics from ample independent trials. Manuscripts may be returned at the editor's discretion for the completion of any lacking or partial statistical data.

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1. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

https://www.jidonline.org/article/S0022-202X(23)02306-0/fulltext

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

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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: Partly

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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?

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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: Major concerns

Authors constructed hair-follicle associated pluripotent (HAP)-cell-sheet for implantation to enhance wound healing in diabetic db/db mice. However, authors missed several critical factors.

1. The cell sheet contains a variety of cells, but which cell plays the critical role is unknown.

2. There was no significant difference in histological results. Moreover, the epidermis and dermis in HAP group were thicker, which was not favorable for tissue regeneration.

3. Why could CD34-positive cells represent neovascularization, does the cell sheet still express CD34 after cell differentiation?

4.What is the biological role of CD34?

Minor concerns

1. The grammar needs to be improved thoroughly over the manuscript.

Reviewer #2: This study entitled 'Hair-follicle associated pluripotent (HAP)-cell-sheet implantation enhanced wound healing in diabetic db/db mice' focuses on utilizing HAP cells derived from hair follicles for treating excisional wound in diabetic conditions. The manuscript requires further refinement before it can satisfy the standards for publication.

major:

1. Concerning the use of flow cytometry for cell characterization from the HAP sheet: a) There appears to be no use of isotype controls in the process. b) CD31 should be stained to exclude endothelial cells. c) In Figure 2A, there are discrepancies in SSC-A (cell granularity) on the y-axis across the four charts. If these cells are from the same batch, uniformity is expected. An explanation for this variation is needed.

2. Regarding the transplantation of 5 × 10^5 cells for a 1 cm circular wound, I am skeptical about the effectiveness. Has there been any comparison with other cell types, such as keratinocytes? How many cells are typically implanted? Additionally, why is this particular type of cell, which requires a month to culture, considered more effective?

3. The claim that the HAP-cell-sheet accelerates wound healing is not convincingly supported by the presented results: a) The sheet does not seem to properly align with the wound shape. b) Re-epithelialization is crucial for wound closure. Has there been an assessment of when re-epithelialization occurs in db/db mice, particularly whether it happens sooner in the HAP-sheet treated group compared to the control group? It would be beneficial to perform Krt14 staining and H&E staining around the time of re-epithelialization. c) The survival duration of the transplanted cells in relation to wound healing is uncertain. It's important to demonstrate the presence of transplanted cells throughout the healing process.

minor:

1. The structure of the article could be improved for better clarity. I suggest organizing it as follows: a) Isolation and characterization of HAP sheet cells. b) The role of HAP-cell-sheets in accelerating wound healing. c) The influence of HAP-cell-sheets on fibroblast growth and VEGF accumulation. d) The impact of HAP-cell-sheets on promoting angiogenesis. e) How HAP-cell-sheets may reduce inflammation.

2. The process of isolating HAP is not sufficiently detailed: a) What methods are employed to prevent contamination? b) Regarding lines 91-93, are the plates coated with Matrigel that detaches along with the cells? If Matrigel is part of the sheet, consider including a control group using only Matrigel sheets in both the wound scratch assay and in vivo healing studies. If there's no Matrigel, please clarify how the liquid cell pellet is maintained at the wound's center. c) Over the course of the three-week culture period, how frequently was the medium changed?

3. In Figure 2A, it would be beneficial to include the gating strategy used in the flow cytometry analysis.

4. Concerning line 251, the statement "CD34-positive cells were observed around the follicles (Fig 2B)" needs clarification. What is the intended message here?

5. In Figure 7A, please specify which section of the wound is being shown.

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Reviewer #1: No

Reviewer #2: No

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Hair-follicle associated pluripotent (HAP)-cell-sheet　implantation enhanced wound healing in diabetic db/db mice

PONE-D-23-39123R1

Dear Dr. Hasegawa-Haruki,

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,

Jimin Han

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Article ready to accept.

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 #3: All comments have been addressed

Reviewer #4: 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 #3: Yes

Reviewer #4: Yes

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

Reviewer #3: Yes

Reviewer #4: Yes

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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 #3: Yes

Reviewer #4: 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 #3: Yes

Reviewer #4: Yes

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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 #3: The manuscript has been revised to address all the errors identified in the reviewer's comments. I have carefully reviewed the changes and agree to the publication of the article.

Reviewer #4: (No Response)

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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 #3: No

Reviewer #4: No

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