Dear Dr. Schlaeger,

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.

• 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,

Francesco Bertolini, MD, PhD

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2.  Please update your submission to use the PLOS LaTeX template. The template and more information on our requirements for LaTeX submissions can be found at http://journals.plos.org/plosone/s/latex .

3. Thank you for submitting your work to PLOS ONE. We note that you have not mentioned the source imMKCL cell lines used in this study. Before we can continue with your submission, kindly mention the details in the Method section. Thank you for your attention to this request. We look forward to hearing from you.

4. Thank you for stating the following financial disclosure:

“This work was made possible by the generous support of the Boston Children’s Hospital Stem Cell Program, the NIDDK (U54DK110805) and Megakaryon Inc. This work was supported in part by the Core research center for next-generation medicine utilizing Cell and gene therapy grant (23bm1323001h0001 to K.E.) from AMED, the CiRA Foundation Fund (to K.E.), a grant-in-aid for scientific research (KIBAN S, 21H05047, to K.E) from the Japan Society for the Promotion of Science (JSPS), and collaborating project funding from Megakaryon Co. Ltd. (to K.E., A. F., and T. S.).”

Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

5. Thank you for stating the following in the Acknowledgments Section of your manuscript:

“The authors express their gratitude to Dr. Ronald Mathieu and the Boston Children’s Hospital flow cytometry core facility for excellent flow cytometry service and to the members of the George Q. Daley, Leonard I. Zon, Trista North, Andrew L. Frelinger, Martha Sola-Visner, Joseph Italiano, Bruce Furie, and Thorsten M. Schlaeger laboratories, including Arunoday Bhan, for valuable suggestions and  discussions as well as technical assistance. This work was made possible by the generous support of the Boston Children’s Hospital Stem Cell Program, the NIDDK (U54DK110805) and Megakaryon Inc. This work was supported in part by the Core research center for next-generation medicine utilizing Cell and gene therapy grant (23bm1323001h0001 to K.E.) from AMED, the CiRA Foundation Fund (to K.E.), a grant[1]136 in-aid for scientific research (KIBAN S, 21H05047, to K.E) from the Japan Society for the Promotion of Science (JSPS), and collaborating project funding from Megakaryon Co. Ltd. (to K.E., A. F., and T. S.).”

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“This work was made possible by the generous support of the Boston Children’s Hospital Stem Cell Program, the NIDDK (U54DK110805) and Megakaryon Inc. This work was supported in part by the Core research center for next-generation medicine utilizing Cell and gene therapy grant (23bm1323001h0001 to K.E.) from AMED, the CiRA Foundation Fund (to K.E.), a grant-in-aid for scientific research (KIBAN S, 21H05047, to K.E) from the Japan Society for the Promotion of Science (JSPS), and collaborating project funding from Megakaryon Co. Ltd. (to K.E., A. F., and T. S.).”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

6. Thank you for stating the following in the Competing Interests section:

“K.E. is a founder of Megakaryon and a member of its scientific advisory board without salary and receives research funding from Megakaryon, Otsuka Pharmaceutical, and Kyoto Manufacturing Co. T.S. received research funding from Megakaryon. Y.H. and K.F. are currently employed by Otsuka Pharmaceutical CO., Ltd., Osaka, Japan. The remaining authors declare no competing financial interests”

Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf.

7. In the online submission form, you indicated that “All relevant data are within the manuscript and its Supporting Information files. Image processing scripts and raw imaging data from the chemical genetic screen and time-course imaging experiments are available upon request (corresponding author)”

All PLOS journals now require all data underlying the findings described in their manuscript to be freely available to other researchers, either 1. In a public repository, 2. Within the manuscript itself, or 3. Uploaded as supplementary information.

This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If your data cannot be made publicly available for ethical or legal reasons (e.g., public availability would compromise patient privacy), please explain your reasons on resubmission and your exemption request will be escalated for approval.

8. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

9. We notice that your supplementary figures are uploaded with the file type 'Figure'. Please amend the file type to 'Supporting Information'. Please ensure that each Supporting Information file has a legend listed in the manuscript after the references list.

10. We notice that your supplementary tables are included in the manuscript file. Please remove them and upload them with the file type 'Supporting Information'. Please ensure that each Supporting Information file has a legend listed in the manuscript after the references list.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: No

Reviewer #2: Yes

**********

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: No

**********

Reviewer #1: The study by Nakamura et al. investigated the maturation and platelet generation capacity of immortalized megakaryocyte-like cells (imMKCLs) using single-cell time-course imaging and a chemical genetics screen. The authors identified microtubule-destabilizing agents, such as vincristine (VCR), as enhancers of proplatelet formation, which reduced microtubule content in megakaryocytes, similar to turbulence. These findings suggest that reduction in microtubules could be a central trigger for proplatelet formation. While the VCR-treated platelets lacked the characteristic marginal band, they remained functional, as demonstrated by their response to agonists and ability to restore haemostasis in an in vivo thrombocytopenia model.

The manuscript presents an original, interesting and well-performed study, but there are some inconsistencies, missing quantifications, and areas requiring clarification.

The results describe the maturation process as "fast," yet the methods indicate a six-day maturation period (D0-D6). It would be helpful to use more precise language to describe the maturation timeline. Do the authors observe terminally mature cells already at D1? Additionally, it is unclear when imaging was initiated during this process, whether it began at the start of maturation (D0), after some days of differentiation, or exclusively on D6. It is also unclear whether shaking conditions were maintained during the 45-hour imaging period, what speed of shaking was applied, and whether between D0-D6 the cells were further diluted to maintain a specific cell density or if the media was replaced at any point during the differentiation process. Clarifying these conditions would be important for interpreting the asynchronous platelet production observed. Furthermore, there is an inconsistency between the stated dimensions of the microwells in the figure legend (200x200x100 μm) and the methods section (200x200x200 μm).

In addition, the authors should provide quantification, which is likely already available to them: What percentage of cells released less than 10 or over 50 PLPs? How does the size of the cells correlate with PLP formation activity, do larger cells release more PLPs, or is there no correlation? Additionally, how homogeneous are the cell diameters in these experiments? The authors should provide details on the percentage of cells that fall into size categories such as smaller than 15 μm, between 15-25 μm, and 25-35 μm or larger. While the authors state in the Fig. 1 legend that both PLP formation activity and MK maturation are heterogeneous, they do not provide quantitative data supporting the second claim. Including these analyses would provide valuable insight into the observed variability.

The results in Fig. 2D are indeed impressive, however the statement “these results demonstrate the effectiveness of our chemical genetics screening approach in identifying compounds … that affect imMKCL terminal differentiation” may be overstated. While the results suggest that these compounds increase PPF, it is unclear if they also enhance megakaryocyte differentiation. Ploidy or cell size are not explicitly reported and should be assessed to substantiate claims. It would be also valuable to test whether the observed effects are specific to prolonged treatment or if adding the compounds only during the last 24 hours yields similar outcomes. Statistical analysis for Fig. 2D is missing and should be included. Could the authors repeat the single-cell time course confocal imaging studies (Fig. 1B) to determine whether VCR increases the percentage of cells generating proplatelets or even enhance PLP production beyond the levels of the highest-producing control cells? Additionally, the Fig. S1B legend contains an incomplete sentence, and the concentration unit is missing in S1E.

In Fig. 3D, the images show smaller cells under turbulence conditions, with increased numbers of naked nuclei and F-actin-positive structures. Could the authors clarify why cells are smaller at earlier time points under turbulence conditions? If turbulence enhances maturation, one might expect larger cells. The authors should discuss whether the observed changes reflect physical fragmentation or active maturation, as the flow cytometry plot suggests the latter, and speculate on the potential mechanism driving this process. Is Fig. 3B gated on the larger cells, or does it represent the signal from all cells? It would be beneficial to duplicate this plot separately for MKs and PLPs to clarify the contributions of each population. Additionally, could the authors provide quantification for Fig. 3D to support the visual observations?

Both turbulence (Fig. 3D) and VCR (Fig. 4E) appear to reduce microtubule levels, which could suggest that combining these conditions might not lead to further improvements in platelet yield. Did the authors test this scenario?

The authors should directly acknowledge the complete lack of the marginal band in VCR-treated platelets when describing Fig. S3B. This is a critical observation that should be transparently reported, as it has significant implications for platelet function, particularly in processes dependent on the marginal band. The authors could also consider testing their platelets in assays where microtubules play a central role, such as spreading, shape change, and contractility, to further support their claim that platelets generated with VCR show increased production without compromising function. Alternatively, they could refine their claims by clarifying that while the platelets performed comparably to controls in the assays tested, not all relevant functions were evaluated, and highlight in the discussion that microtubule-dependent processes could potentially be affected by VCR treatment.

The nature of the statistical test is only described for Fig. 5C. The authors should clarify the specific statistical tests used for each subpanel where statistics were applied to ensure transparency and reproducibility.

Reviewer #2: Enhancing the effectiveness of producing platelets from megakaryocytes (MKs) in vitro presents a major challenge and is of great importance. In this manuscript, Nakamura et al. performed high-throughput screening and identified microtubule (MT) destabilizing agents, including the vinca alkaloid vinblastine and vincristine (VCR), as potential promoters of imMKCL maturation and proplatelet formation. The overall study is interesting and significant for improving platelet yield from MKs. However, additional evidence is necessary to substantiate the claims made by the authors.

Major concerns�

1. The authors found that imMKCLs sampled from culture systems with turbulent flow (turbulence+) showed reduced microtubule staining in terminally maturing imMKCLs compared to those sampled from static culture conditions (turbulence-), especially after day 3 (Fig 3D), similar to the expected phenotype of administering VCR. And the platelet yield per MK from the turbulence+ group is much higher than turbulence- group. How does the platelet yield per MK compare between the VCR treatment and the turbulence+ group? Can chemical compounds effectively replace the complex bioreactor devices?

2. The authors compared surface-expression of P-Selectin/CD62P and PAC1 between control platelets and platelets activated with the platelet agonists ADP and TRAP-6. Since platelet are easily activated during the culturing and experimental processing, please provide the data for P-Selectin/CD62P and PAC1 expression without stimulation by any agonist.

3. After transfused into mice, the circulation of VCR-iPSC-PLTs from both VCR treatment groups was significantly decreased compared to control iPSC-PLTs at all time points up to 24 hours. What is the possible mechanism underlying this phenomenon?

4. Although exposing imMKCLs to VCR boosts the yield of iPSC-PLTs, but VCR-iPSC-PLTs show reduced persistence and perform not as well as control iPSC-PLTs, as shown in Fig 5E and S3A. What are the advantages of using VCR in platelet-producing culture systems with turbulent flow?

Minor concerns�

1. The culture condition of the turbulence+ group in Fig 3C is similar to the DMSO group in Fig 4A, but the platelet yield per MK of the DMSO group is much lower than that of the turbulence+ group. Does DMSO play a role in decreasing platelet yield?

2. Some language inaccuracies are present in the text. Please refine the manuscript.

**********

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

**********

[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.

Association of Microtubule Destabilization With Platelet Yields in Terminally Differentiating hiPSC-derived Megakaryocyte Lines

PONE-D-24-54484R1

Dear Dr. Schlaeger,

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.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager®  and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

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,

Francesco Bertolini, MD, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

**********

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

**********

Reviewer #1: The authors have done a thorough and commendable job in addressing the concerns raised during the first round of review. All comments have been adequately addressed, and the revised manuscript has been significantly improved. This reviewer has no further concerns.

Reviewer #3: The authors have adequately addressed the reviewers’ concerns. The paper is now suitable for publication.

**********

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 #3: No

**********