Toward a better definition of hematopoietic progenitors suitable for B cell differentiation

Florian Dubois; Anne Gaignerie; Léa Flippe; Jean-Marie Heslan; Laurent Tesson; Mélanie Chesneau; Fabienne Haspot; Sophie Conchon; Laurent David; Sophie Brouard

doi:10.1371/journal.pone.0243769

Peer Review History

Original SubmissionJuly 23, 2020
27 Aug 2020 Decision Letter - Xiaoping Bao, Editor PONE-D-20-22523 Toward a better definition of hematopoietic progenitors suitable for B cell differentiation PLOS ONE Dear Dr. Dubois, 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. The reviewers are concerned about the sample sizes and B cell characterization. Please address the comments accordingly during the revision. Please submit your revised manuscript by Oct 11 2020 11:59PM. 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. 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[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? 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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: No ****** 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 ****** 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 ****** 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 authors compared CD34+ progenitors derived from cord blood and human ESC. They identified CD31(int)CD45(int) as efficient hematopoietic progenitors suitable for B cell differentiation. Interestingly, human PSCs are less efficient to generate B cells than cord blood stem cells. Major concerns: 1. In many experiments or conditions, there is only 1 independent experiment and there are no solid statistical comparison to reach any conclusion. 2. In the first 10 days derivation of CD34+ cell from hPSCs, although the authors tried to play with different concentrations or combinations of cytokines to get the highest efficiency, the transition from hPSCs into specific mesoderm progenitors and then differentiate into CD34+ progenitors are overlooked. Certain mesoderm progenitors (i.e., anterior or posterior) favor the hematopoietic differentiation. 3. The authors discussed a lot of efficienty difference and the functional characterization of the differentiated B cells except for B cell markers is missing Reviewer #2: In this manuscript, Florian Dubois et al. reported that they were interested in the relationship between B cells and hPSCs-derived hematopoietic stem cells. They differentiated the hPSCs into hematopoietic stem cells through the OP9 stromal cell, and the B cells were generated on MS-5 stromal cells. They investigated the influence of various cytokine conditions on the hematopoietic stem cell generation, and they highlight CD31intCD45int phenotype as a possible marker of hematopoietic progenitors suitable for B cell differentiation. This manuscript should be revised as below. Major modification: (1) At the introduction part, they authors should make more illustration about the relationship between B cells and hematopoietic stem cells, especially the published protocols for B cell differentiation. (2) At the experiment section, the OP9 cells were cultured in αMEM medium instead of aMEM medium. (3) The authors should perform further characterization on the iPSC-derived hematopoietic stem/progenitor cells, the characterization is not enough. (4) As a biological experiment data, the authors should make additional statistical analysis, for example, Fig 2-7. (5) In order to make this manuscript better, the authors should improve the layout quality of the Figures, especially Fig 5. (6) The authors should make a scheme illustration to present their key ideas or results. ****** 6. 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:** Yun Chang [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. https://doi.org/10.1371/journal.pone.0243769.r001
Revision 1
12 Nov 2020 Author Response Reviewer #1: The authors compared CD34+ progenitors derived from cord blood and human ESC. They identified CD31(int)CD45(int) as efficient hematopoietic progenitors suitable for B cell differentiation. Interestingly, human PSCs are less efficient to generate B cells than cord blood stem cells. Major concerns: 1. In many experiments or conditions, there is only 1 independent experiment and there are no solid statistical comparison to reach any conclusion. For more clarity, we chose to only show the experiments performed with one cytokine concentration of 10ng/mL in Figure 2B and 2C. Nevertheless, this experiment with the condition D was reproduced several times with cytokines used at 25ng/mL (Figure 3A, n=3). As mentioned by reviewer 2, we performed statistical tests and confirmed statistical difference between the conditions and the CTRL in the figure 2, 3 and S1. 2. In the first 10 days derivation of CD34+ cell from hPSCs, although the authors tried to play with different concentrations or combinations of cytokines to get the highest efficiency, the transition from hPSCs into specific mesoderm progenitors and then differentiate into CD34+ progenitors are overlooked. Certain mesoderm progenitors (i.e., anterior or posterior) favor the hematopoietic differentiation. Regarding OP9 stromal cells favor hematopoietic differentiation, we only focus about the hematopoietic progenitor generation. Nevertheless, we will look forward to check if there is a lack of mesoderm progenitors susceptible for hematopoietic differentiation in our model. 3. The authors discussed a lot of efficienty difference and the functional characterization of the differentiated B cells except for B cell markers is missing During our differentiation, we show that hematopoietic progenitors follow the specific stage-differentiation of B cells both in term of molecular and transcriptional marker. However, the few amount of B cells generated did not permit any sorting and functional characterisation, we first need to optimise the protocol and reach a sufficient number of B cells. Reviewer #2: In this manuscript, Florian Dubois et al. reported that they were interested in the relationship between B cells and hPSCs-derived hematopoietic stem cells. They differentiated the hPSCs into hematopoietic stem cells through the OP9stromal cell, and the B cells were generated on MS-5 stromal cells. They investigated the influence of various cytokine conditions on the hematopoietic stem cell generation, and they highlight CD31intCD45int phenotype as a possible marker of hematopoietic progenitors suitable for B cell differentiation. This manuscript should be revised as below. Major modification: (1) At the introduction part, they authors should make more illustration about the relationship between B cells and hematopoietic stem cells, especially the published protocols for B cell differentiation. Reviewer 2 is right, we highlighted this point in the introduction section (l.50). (2) At the experiment section, the OP9 cells were cultured in αMEM medium instead of aMEM medium. We double-checked and we confirmed that OP9 cells were cultured in the right �MEM medium (without nucleoside) supplemented with fetal bovine serum to a final concentration of 20% as recommended by ATCC; Vodyanick et al., 2005; Carpenter et al., 2011; and French et al., 2015. (3) The authors should perform further characterization on the iPSC-derived hematopoietic stem/progenitor cells, the characterization is not enough. Because of the actual situation due to research shut down during the Covid19 pandemic, we were not able to performed new B cell differentiation. (4) As a biological experiment data, the authors should make additional statistical analysis, for example, Fig 2-7. Reviewer 2 is right, when possible (n > 3), statistical analysis were performed. We thus updated the material and method section (l.138) and the figures 2, 3 and S1. (5) In order to make this manuscript better, the authors should improve the layout quality of the Figures, especially Fig 5. We improved the layout quality of all figures. (6) The authors should make a scheme illustration to present their key ideas or results. We added a scheme illustration of the B cell differentiation key steps and its reference in the discussion section (l.370). Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0243769.r002
30 Nov 2020 Decision Letter - Xiaoping Bao, Editor Toward a better definition of hematopoietic progenitors suitable for B cell differentiation PONE-D-20-22523R1 Dear Dr. Dubois, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, 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, Xiaoping Bao, 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 ******** 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 author addressed my questions and I recommend to publish this manuscript in the journal of plos one. Reviewer #2: The authors have answered all of the questions.This manuscript was in good quality for publication. ****** 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:** YUN CHANG https://doi.org/10.1371/journal.pone.0243769.r003
Formally Accepted
4 Dec 2020 Acceptance Letter - Xiaoping Bao, Editor PONE-D-20-22523R1 Toward a better definition of hematopoietic progenitors suitable for B cell differentiation Dear Dr. Dubois: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Xiaoping Bao Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0243769.r004

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