Human induced pluripotent stem cell-derived three-dimensional cardiomyocyte tissues ameliorate the rat ischemic myocardium by remodeling the extracellular matrix and cardiac protein phenotype

Junya Yokoyama; Shigeru Miyagawa; Takami Akagi; Mitsuru Akashi; Yoshiki Sawa

doi:10.1371/journal.pone.0245571

Peer Review History

Original SubmissionDecember 30, 2020
1 Feb 2021 Decision Letter - Vincenzo Lionetti, Editor PONE-D-20-41017 Human induced pluripotent stem cell-derived three-dimensional cardiomyocyte tissues ameliorate the rat ischemic myocardium by remodeling the extracellular matrix and cardiac protein phenotype PLOS ONE Dear Dr. Sawa, 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. All issues raised by expert reviewers are required. Please submit your revised manuscript by Mar 18 2021 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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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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Vincenzo Lionetti, M.D., 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 [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: No Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 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: This study investigates the beneficial effects of implantation of 3D hiPSC-cardiomyocytes sheets coated with FN and gelatin on the cardiac performance in MI rats. The authors assessed the cardiac function, graft survival and vascularization in control group and iPSC-CM group. Though the study design and the idea behind the 3D FN G coated iPSC-CT is interesting, however, the experimental design needs significant improvement before this study can be considered for publication. Following are my more specific comments. 1)What was the control used in the hypoxia and in vivo study? Page 9, line 134 the authors have mentioned uncoated, is this the right control to be used to compare the layer-by-layer generated iPSC-CT? Generally, for generation of CT one would use collagen, fibronectin or Matrigel? 2)Generally, in case of the cardiomyocytes maintained in media containing FBS, the FBS tend to prevent the beating of cells. Authors need to clarify this, how they were able to get beating cardiomyocytes for the experiment during transplantation? 3)What is the conformation test for the coating of fibronectin and gelatin on iPSC-CMs? Authors must perform analytical evaluations similar to SEM and FTIR and provide data to improve rigor of the current study. 4)There is no sham group in the experiment setup. Authors should include sham group during the analysis of the in vivo data. 5)Page 6 line 86 replace iPSC-CM as iPSC. Reviewer #2: This manuscript has valuable information to scientists who involve in research of heart failure.Especially, as compared to other multipotent stem cells, such as ES cell, iPS cell has potential benefit by minimizing the risk of rejection, ethical problem, and tumorigenesis. It seems that creating iPS cell still has comlicated process that has to be improved, I think prompt clinical trial should be recommended. ****** 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: 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. https://doi.org/10.1371/journal.pone.0245571.r001
Revision 1
19 Feb 2021 Author Response Reviewer 1: Q1. What was the control used in the hypoxia and in vivo study? Page 9, line 134 the authors have mentioned uncoated, is this the right control to be used to compare the layer-by-layer generated iPSC-CT? Generally, for generation of CT one would use collagen, fibronectin or Matrigel? A1. Response: Thank you for your comment. The FN/G coated cells and uncoated cells were compared in the hypoxia experimental system. In vitro, when 3 × 106 cells were placed in a 24-well cell culture insert without coating, tissues could not be created as the cells became heterogeneous. Thus, we were not able to compare coated and uncoated 3D-hiPSC-CTs. Additionally, when 3D-hiPSC-CTs were generated, we used only fibronectin and gelatin—not collagen—as a small population of vimentin-positive cells are found in iPS induction; there is a possibility that collagen derived from fibroblasts may be present. Q2. Generally, in case of the cardiomyocytes maintained in media containing FBS, the FBS tend to prevent the beating of cells. Authors need to clarify this, how they were able to get beating cardiomyocytes for the experiment during transplantation? A2. Response: Thank you for pointing this out. In this study, 3D-iPS-CTs were cultured in a medium containing 10% FBS serum. We confirmed that the cardiomyocytes were beating in this experimental system daily and that the myocardial tissue itself was beating at the time of transplantation. FBS was flushed out avoid immunological rejection prior to CT transplantation. We have included the relevant sentences in the manuscript. Q3. What is the conformation test for the coating of fibronectin and gelatin on iPSC-CMs? Authors must perform analytical evaluations similar to SEM and FTIR and provide data to improve rigor of the current study. A3. Response: We appreciate the helpful suggestion. As suggested, we have presented the TEM image. We have included the following: “Ultrastructural analysis of 3D-hiPSC-CT using transmission electron microscopy. In the 3D-hiPSC-CT, irregularly shaped hiPSC-CMs aggregate in several layers. Each manufactured cardiomyocyte contained some myofibrils and mitochondria with a few cristae. The cells contacted each other via desmosomes (Fig 5A). The implanted 3D-hiPS-CT showed clear thickened myofibrils, Z-band, and mitochondria with many lamellar cristae. The adherens junction and desmosome were also disclosed between the iPSC-CM (Fig 5B).” In regards to the conformation test for coating, the iPS-CM was coated by the method by our co-author Akashi [1], which confirmed coating; thus, we had not confirmed the results of coating again. [1] Nishiguchi A, Matsusaki M, Miyagawa S, Sawa Y, Akashi M. Dynamic nano-interfaces enable harvesting of functional 3D-engineered tissues. Adv Healthc Mater. 2015;4(8): 1164-1168. doi:10.1002/adhm.201500065, PubMed: 25728509. Q4. There is no sham group in the experiment setup. Authors should include sham group during the analysis of the in vivo data. A4. Response: In vitro, when 3 × 106 cells were placed in a 24-well cell culture insert without coating, tissues could not be created as the cells became heterogeneous. Thus, the transplant experiment was a comparative experiment between sham control—which had no transplanted tissue—and the 3D-hiPS CT transplant. Q5. Page 6 line 86 replace iPSC-CM as iPSC. A5. Response: Thank you, as suggested we have revised iPSC-CM to iPSC. Reviewer2: Thank you for your peer review despite your busyness. We will continue to strive for the development of the field of regenerative medicine. Attachments Attachment Submitted filename: revise.docx https://doi.org/10.1371/journal.pone.0245571.r002
23 Feb 2021 Decision Letter - Vincenzo Lionetti, Editor Human induced pluripotent stem cell-derived three-dimensional cardiomyocyte tissues ameliorate the rat ischemic myocardium by remodeling the extracellular matrix and cardiac protein phenotype PONE-D-20-41017R1 Dear Dr. Sawa, 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, Vincenzo Lionetti, M.D., PhD 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: No 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: Authors have addressed all my concerns. The manuscript has improved significantly. I have no further comments comments. Reviewer #2: I think asuthors have addressed all the required change suggested in previous review. I would like to encourage iPS study to be available in real clinical setting soon. ****** 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 https://doi.org/10.1371/journal.pone.0245571.r003
Formally Accepted
5 Mar 2021 Acceptance Letter - Vincenzo Lionetti, Editor PONE-D-20-41017R1 Human induced pluripotent stem cell-derived three-dimensional cardiomyocyte tissues ameliorate the rat ischemic myocardium by remodeling the extracellular matrix and cardiac protein phenotype Dear Dr. Sawa: 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 Prof. Vincenzo Lionetti Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0245571.r004

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