PONE-D-19-33452

The Evaluation of a Tissue-Engineered Cardiac Patch Seeded with hiPS Derived Cardiac Progenitor Cells in a Rat Left Ventricular Model

PLOS ONE

Dear Dr. Shinoka,

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Academic Editor

PLOS ONE

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"All animals received humane care in compliance with the National Institutes of Health (NIH) guideline for the Care and Use of Laboratory Animals. The Institutional Animal Care and Use Committee at Nationwide Children’s Hospital approved the use of animals and procedures described in this study(AR12-00079). Adult male nude athymic rats (Jackson Laboratories, Bar Harbor, ME) weighing 230–300 g were used for the left ventricle wall replacement procedure. General anesthesia was induced with ketamine (50 mg/kg, i.p) and xylazine (5 mg/kg, i.p); anesthesia was maintained using isoflurane (1.5%) in oxygen. Animals were intubated with a 16-gauge catheter and respiration was maintained at 60 cycles per minute with a tidal volume of 2.5 ml. ".

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[This work was supported by in part by a grant from the National Institutes of Health

(R01- HL098228 to Dr. Breuer). Dr. Shinoka receives grant support from Gunze Ltd.

(Kyoto, Japan). The is study was performed by the authors, who also had full control

of the study design, methods used, outcome measurements, data analysis, and

production of the written report.].

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 [Dr. Matsuzaki was the recipient of funding award from Uehara Memorial Foundation (Tokyo Japan) in 2019

This work was supported by in part by a grant from the National Institutes of Health (R01- HL098228 to Dr. Breuer). Dr. Shinoka receives grant support from Gunze Ltd. (Kyoto, Japan). The is study was performed by the authors, who also had full control of the study design, methods used, outcome measurements, data analysis, and production of the written report.

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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

Reviewer #3: Partly

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

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: No

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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

Reviewer #3: 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

Reviewer #3: 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: Matsuzaki and colleagues submit their manuscript entitled, “The Evaluation of a Tissue-Engineered Cardiac Patch Seeded with hiPS Derived Cardiac Progenitor Cells in a Rat Left Ventricular Model” for consideration of publication in PLOS ONE. The authors use a rodent model to test whether a cell-seeded biodegradable tissue-engineered patch provides beneficial effects in LV function. The experiments are well designed with appropriate controls. Unfortunately, but not surprising cell-seeding of iPS cells did not lead to an increase in cardiomyocytes. The authors do demonstrate that a biodegradable patch can be used as a viable option for ventricular rupture.

Comments

1. The potential benefit of the biodegradable patch was the ability to cell-seed the patch. However, since the cell-seeding did not lead to increased myocardial cells, authors should clarify the benefit of their patch over non-absorbable patches.

2. Inclusion of a non-biodegradable patch as a comparison to the biodegradable patch could have been included. Do the authors have any comparison data? Especially in light that at 36 weeks, there was no difference between cell-seeded and no cell-seeding. So is a biodegradable patch add benefit?

3. Was vascularization assessed? Also, was staining performed for fibroblasts?

Reviewer #2: The work described in this manuscript addresses an important issue of designing a cell-seeded cardiac patch to improve cardiac function. Successful development of such a system could improve cardiac function in adults after ventricular rupture. The limited results in this study show that biodegradable patches implanted at the left ventricular apex with or without cardiomyocytes did not improve cardiac function. The reason for this lack of effect could not be clearly identified due to limitations of the study design.

The authors provide no assessment of cardiomyocyte function or organization prior to implantation, so it unclear whether the implanted cardiomyocytes had any contractile function at the time of implantation.

The small sample size in each group (1 and 2 month time points had 2 animals in each group and the 6 and 9 each had 3 animals in each group) precluded detection of differences between the two groups except at 8 weeks for ejection fraction. The sample size is much smaller than the authors used in their prior study, reference 11.

The histological analysis of the implants did not examine for the presence of cardiomyocytes or vascularization in the patches. Thus, the reason for the difference in ejection fraction due to cell seeding could not be evaluated.

Specific Comments

1. Figure 4. Horizontal axis label is needed for each panel.

2. Figure 4. Were the changes in LV systolic and diastolic volumes over time significant? The statistical methods used indicate that t-tests were used, but the results in Figure 4 indicate that a two-factor ANOVA (time and treatment) should be used to evaluate trends with time.

Reviewer #3: This study tested the feasibility of applying stem-cell patches with a biodegradable substrate to damaged ventricular tissue to enhance long-term remodeling. Engineered tissues for cardiac repair are evolving and in-vivo studies such as are valuable results that may help the field advance toward human use.

Although the goals of the study are modest, and methods used to quantify histological changes are limited, the results do provide useful information and show that this type of tissue engineered patch with human iPS cells and some type of biodegradable substrate can integrate with the native myocardium, albeit with limited functional outcomes at the present time.

A major limitation of the study is that the application of these patches, as stated in the introduction, can help with tissue remodeling in situations such as ventricular aneurysm, but the studies were done in a much different ventricular trauma injury model. The acute injury response to the procedure here is much different than the human diseases mentioned in the introduction and as rationale for the study. This difference is not discussed in the paper.

The methods should describe how the microscopic imaging were analyzed and what exactly was quantified, including sample sizes. Also, information on orientation of the sections, since cardiac and scar tissue, the patch and the geometry of the wall are all have direction-dependence structure. Finally, better quantification of the cell numbers and degree of calcification should be done in addition to the qualitative descriptions in the results.

Part of the study is the examine the biodegradability of the patch substrate, but this was not quantified, and only vaguely described in the paper. How do we know the that “some patch material still remains and is covered by collagen” or that it actually degrades? Some kind measure to back up these statements would be useful.

The quantitative results are likely under-powered, with small sample sizes (n=2 or 3). Have the authors done any power analysis or other statistical approaches to determine if the numerical results (echo studies) are actually statistically valid? Changes in LV function is one of the stated goals of the study, and it is hard to interpret the functional results with these sample sizes.

The conclusions paragraph can be improved. The first and third sentences are not conclusions. The second sentence is the only actual statement of a conclusion, and certainly does not summarize the main findings of the paper.

Minor:

Please include page numbers for easier referencing. Also, please use indentation or spacing for new paragraphs.

Abstract, last sentence: “differentiate into cardiomyocytes survive”, correct wording.

Intro, line 4: “cell attachment or migration” migration of what cell type?

Intro, line 14: “Litations”, Limitations??

The discussion starts with references to post-infarction remodeling, but the current study does not use that model. It would be better for the rationale for the current experiments to be better related to the same type of remodeling that would occur with the current animal model. Why is “infection” mentioned, when this is not part of the current study (but durability and biocompatibility are)?

Discussion, first para: “sustain regular rhythm”, why mention this is such a short “intro” in the discussion, when it is not part of the current study?

Discussion: “In the past, heart tissue was considered non-renewable and the heart was considered the final organ”. Wording is awkward.

Discussion: “….to create a pulsation model.” Not clear what this type of model refers to.

Discussion: “and host species leading to a non-sychronized heartbeat” not-clear that this is the case or that this would happen here. Also typo in “synchronized”

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Reviewer #1: No

Reviewer #2: Yes: George A. Truskey

Reviewer #3: No

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Attachments

Attachment

Submitted filename: Matsuzaki_patch 122219.docx

PONE-D-19-33452R1

The Evaluation of a Tissue-Engineered Cardiac Patch Seeded with hiPS Derived Cardiac Progenitor Cells in a Rat Left Ventricular Model

PLOS ONE

Dear Dr. Shinoka,

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.

We would appreciate receiving your revised manuscript by Jun 08 2020 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

YIRU GUO, M.D., F.A.H.A.

Academic Editor

PLOS ONE

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

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: (No Response)

Reviewer #2: 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?

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

Reviewer #3: (No Response)

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

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

Reviewer #3: (No Response)

**********

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

Reviewer #3: (No Response)

**********

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: Matsuzaki and colleagues submit their revised manuscript for evaluation for publication in PlosOne. The initial criticism which dampened this reviewer’s enthusiasm for the manuscript was the negative results that cell-seeding of iPS cells did not lead to an increase in cardiomyocytes nor longer term improvement of cardiac function. Additionally, the lack of an adequate control for the biodegradable patch was excluded. The authors have only made minimal changes to the manuscript in the discussion highlighting the negative results, but more importantly not providing adequate discussion of the potential positive effects of the biodegradable patch without cell seeding. There is a an abundance of literature of the use of bioactive scaffolds without the use of cell transplantation for myocardial repair/regeneration.

Reviewer #2: While preliminary, the results show that merely adding the iPS-derived cardiac progenitor cells is insufficient to ensure the survival of these cells in a cardiac patch. This information is worth transmitting to the research community. Study limitations are duly noted.

Reviewer #3: (No Response)

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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.

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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 #1: No

Reviewer #2: No

Reviewer #3: 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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

The Evaluation of a Tissue-Engineered Cardiac Patch Seeded with hiPS Derived Cardiac Progenitor Cells in a Rat Left Ventricular Model

PONE-D-19-33452R2

Dear Dr. Shinoka,

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,

YIRU GUO, M.D., F.A.H.A.

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

**********

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

**********

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

Reviewer #1: 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

**********

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

**********

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 addressed concerns. No further comments or revisions needed. Although a negative study, the manuscript further provides evidence that iPS cells alone are not beneficial for myocardial repair and that a polymer alone may impact LV remodeling after an injury.

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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.

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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 #1: No