Treatment of corneal endothelial damage in a rabbit model with a bioengineered graft using human decellularized corneal lamina and cultured human corneal endothelium

Francisco Arnalich-Montiel; Adrian Moratilla; Sherezade Fuentes-Julián; Veronica Aparicio; Marta Cadenas Martin; Gary Peh; Jodhbir S. Mehta; Khadijah Adnan; Laura Porrua; Ane Pérez-Sarriegui; Maria P. De Miguel

doi:10.1371/journal.pone.0225480

Peer Review History

Original SubmissionMay 24, 2019
21 Aug 2019 Decision Letter - Che J. Connon, Editor PONE-D-19-14728 Treatment of corneal endothelial damage in a rabbit model with a bioengineered graft using human decellularized corneal lamina and cultured human corneal endothelium PLOS ONE Dear Dr De Miguel, 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. I believe the reviewers comments to be pertinent. Proof that the transplanted human cells have remained in place and at a similar number following transplantation for the 4 weeks should be provided. I appreciate that PCR against human DNA was included but as the reviewer indicates this does not give a readout on how many cells were retained. You might argue that the control did not show the same effect so it must be the presence of the human cells. However, the presence of the human cells might stimulate the endogenous cells to migrate/proliferate more aggressively. Perhaps the inclusion of in vivo endothelial cell morphology at multiple time points could help (i.e. at least every 7 days or more). This way one might follow changes in cell morphology or not, the latter suggesting little change. Otherwise immunohistology against human antibodies or preferably Fluorescence in situ hybridization (FISH) using day 30 postmortem corneas. Otherwise I found the article to well written and scientifically robust. If you can address the above concern I support a resubmission of this study ============================== We would appreciate receiving your revised manuscript by Oct 05 2019 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. 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The PLOS ONE style templates can be found at http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 1. Please move Fig. 2 to a Supporting Information file 2. In your Methods section, please provide additional information regarding the donated tissues or organs used in the study. Please specify where the tissues come from. Please also specify whether the study involved the use of donated tissue/organs from any vulnerable populations. Examples of vulnerable populations include prisoners, subjects with reduced mental capacity due to illness or age, and children. If such a population was used, please ensure you have describe the population and justify the decision to use tissue/organ donations from this group. If not, please state in your Ethics Statement, 'None of the transplant donors were from a vulnerable population and all donors or next of kin provided written informed consent that was freely given. 3. 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. [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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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: Authors used human decellularized corneal stroma as a career of bioengineered corneal graft and performed experiments in rabbit eyes. They concluded the experimental graft was effective in improving visual quality. However, the methods used here did not prove and support their conclusions. Experimentally proliferated hCECs might not have enough pumping function. They need to prove it either in vitro or in vivo. Rabbit endothelial cells regenerate very fast. 0.5mm gap between graft and host endothelial edge could be filled out quickly, probably within the observational period in this study. Authors need to show the improvement of the corneal edema/clarity was due to human corneal cells seeded, not from regenerated rabbit healthy cells. They only performed HE stain of the extracted grafts and did not show the endothelial cells covering the graft were of human origin. Using simple PCR indirectly proves the human cell presence but did not prove the full coverage and survival of hCECs seeded on the graft. Authors measured corneal thickness in HE sections but it must be directly measured in vivo by pachymeter or AS-OCT. ****** 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 [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. https://doi.org/10.1371/journal.pone.0225480.r001
Revision 1
29 Oct 2019 Author Response PONE-D-19-14728 Treatment of corneal endothelial damage in a rabbit model with a bioengineered graft using human decellularized corneal lamina and cultured human corneal endothelium PLOS ONE Answers to editor and Reviewer´s comments: Editors´ Comments I believe the reviewers comments to be pertinent. Proof that the transplanted human cells have remained in place and at a similar number following transplantation for the 4 weeks should be provided. I appreciate that PCR against human DNA was included but as the reviewer indicates this does not give a readout on how many cells were retained. You might argue that the control did not show the same effect so it must be the presence of the human cells. However, the presence of the human cells might stimulate the endogenous cells to migrate/proliferate more aggressively. Perhaps the inclusion of in vivo endothelial cell morphology at multiple time points could help (i.e. at least every 7 days or more). This way one might follow changes in cell morphology or not, the latter suggesting little change. Otherwise immunohistology against human antibodies or preferably Fluorescence in situ hybridization (FISH) using day 30 postmortem corneas. We have performed anti-human nuclei and anti-human mitochondria immunohistochemistries in sections of 30 day postmortem experimental rabbits corneas to show that in fact all endothelial cells are of human origin. This is now shown now in Figure 4A. Otherwise I found the article to well written and scientifically robust. If you can address the above concern I support a resubmission of this study Thank you very much for your kind words, hopefully now we have addressed the downfalls of our article properly. 1. Please move Fig. 2 to a Supporting Information file Done, it is now labelled as SupplFigure1. 2. In your Methods section, please provide additional information regarding the donated tissues or organs used in the study. Please specify where the tissues come from. Please also specify whether the study involved the use of donated tissue/organs from any vulnerable populations. Examples of vulnerable populations include prisoners, subjects with reduced mental capacity due to illness or age, and children. If such a population was used, please ensure you have describe the population and justify the decision to use tissue/organ donations from this group. If not, please state in your Ethics Statement, 'None of the transplant donors were from a vulnerable population and all donors or next of kin provided written informed consent that was freely given. None of the transplant donors were from a vulnerable population and all donors or next of kin provided written informed consent that was freely given. This has been revised and edited in the Materials and Methods manuscript´s section. In addition, a table with all information of the donated tissues has been included (Table 1). 3. 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. This has been now shown in Suppl.Figure2 Reviewers' comments: 5. Reviewer #1: Comments to the Author Authors used human decellularized corneal stroma as a career of bioengineered corneal graft and performed experiments in rabbit eyes. They concluded the experimental graft was effective in improving visual quality. However, the methods used here did not prove and support their conclusions. Experimentally proliferated hCECs might not have enough pumping function. They need to prove it either in vitro or in vivo. We have performed immunohistochemistry demonstrating the pumping function of proliferated hCECs, shown as active Na+/K+-ATPase pump in Figure 1E. We want to thank the reviewer for this comment, as the new data has strengthen considerably our manuscript. Rabbit endothelial cells regenerate very fast. 0.5mm gap between graft and host endothelial edge could be filled out quickly, probably within the observational period in this study. Authors need to show the improvement of the corneal edema/clarity was due to human corneal cells seeded, not from regenerated rabbit healthy cells. They only performed HE stain of the extracted grafts and did not show the endothelial cells covering the graft were of human origin. Using simple PCR indirectly proves the human cell presence but did not prove the full coverage and survival of hCECs seeded on the graft. We agree that the 0.5mm gap could have been filled between the observational period. However, we feel that we have not described our approach very well, as the whole center of the cornea was decellularized by scraping out the endothelial layer, so even if the gap was filled, the majority of the cornea still was devoid of endothelial cells, as demonstrated in the control rabbits. Also that was the reason for a follow up period of only 4 weeks, to ensure not enough time for rabbit endothelial regeneration of the central cornea. Still, we agree that proof of human cell survival and coverage on the graft should be provided, so we have now performed anti-human nuclei and anti-human mitochondria immunohistochemistries in sections of 30 day postmortem experimental rabbits corneas to show that in fact all endothelial cells are of human origin. This is now shown now in Figure 4A. Again we want to thank the reviewer for this most helpful suggestion. Authors measured corneal thickness in HE sections but it must be directly measured in vivo by pachymeter or AS-OCT. We agree that a pachymeter should have been the method of election to measure the corneal thickness, but we could not purchase it from the beginning of the experiment. In fact, we have measures of several but not all rabbits by such method, and have compared such measurements with subsequent ones in HE sections. In all cases, post-fixation measurements were consistently about 85% of the in vivo ones, so we are positive HE measurements are in fact reliable and representative of the in vivo measures when the control and experimental groups are compared, as all of them were processed in the exact same way. Attachments Attachment Submitted filename: ResponsetoReviweresPONE.doc https://doi.org/10.1371/journal.pone.0225480.r002
6 Nov 2019 Decision Letter - Che J. Connon, Editor Treatment of corneal endothelial damage in a rabbit model with a bioengineered graft using human decellularized corneal lamina and cultured human corneal endothelium PONE-D-19-14728R1 Dear Dr. De Miguel, 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. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Che J. Connon Academic Editor PLOS ONE Additional Editor Comments (optional): The main cause of contention has been successfully resolved by inclusion of data in Fig4. Reviewers' comments: None https://doi.org/10.1371/journal.pone.0225480.r003
Formally Accepted
12 Nov 2019 Acceptance Letter - Che J. Connon, Editor PONE-D-19-14728R1 Treatment of corneal endothelial damage in a rabbit model with a bioengineered graft using human decellularized corneal lamina and cultured human corneal endothelium Dear Dr. De Miguel: I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Che J. Connon Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0225480.r004

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