Safety assessment of multiple systemic administration of human mesenchymal stem cell-conditioned medium for various chronic diseases

Norihito Inami

doi:10.1371/journal.pone.0322497

Peer Review History

Original SubmissionJuly 5, 2024
5 Jul 2024 Author Response https://doi.org/10.1371/journal.pone.0322497.r001
20 Aug 2024 Decision Letter - Md Shaifur Rahman, Editor Dear Dr. Inami, 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 mesenchymal stem cell-conditioned medium gives promising results in in vitro and in vivo studies, and it is exciting to see that these mediums can be applied to humans. Therefore, the presented work is valuable, but some requirements must be met in order to be publishable. Introduction 1. The introduction is weak for this study. The importance of using the conditioned medium derived from mesenchymal stem cells for inflammatory diseases should be emphasized. The literature contains many in vitro and in vivo studies on this subject. The introduction should be supported by these studies. Why is it desired to be used in humans? This context should be well established. Methods 2. How many donors were used as sources of mesenchymal stem cells? Was CM prepared separately from the cells obtained from these donors? Which source was the CM used for which disease? It should be specified. 3. Has the content of CM obtained from adipose tissue been compared with that of CM obtained from the umbilical cord? This difference is significant, and the study should have been planned with this in mind. 4. The preparation conditions of CMs should be explained in the method section, such as the number of cells, how long they were kept, how much medium, medium, and supplement content were used, etc. 5. How were the doses determined? Your sample size for INH is 1. This is insufficient. Results 6. What do 1 and 2 represent in Figure 2? The x-axis naming of all graphs should be corrected. Label in an understandable way when looking at the graph or add a description to the figure legend. It is irrational for the "error bar SE" label to be on the x-axis. Add this to the figure legend and label it in a way that makes your data understandable. Discussion 7. The findings should be supported by literature data in the discussion. Possible safety and side effects should be discussed with possible reasons, considering variables such as administration routes, CM source used, disease, etc. Thanks Shaifur Rahman, PhD ============================== Please submit your revised manuscript by Oct 04 2024 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 . 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, Md Shaifur Rahman, Ph.D Academic Editor PLOS ONE Journal requirements: 1. When submitting your revision, we need you to address these additional requirements. 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. We note that you have indicated that there are restrictions to data sharing for this study. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Before we proceed with your manuscript, please address the following prompts: a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially identifying or sensitive patient information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., a Research Ethics Committee or Institutional Review Board, etc.). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories. You also have the option of uploading the data as Supporting Information files, but we would recommend depositing data directly to a data repository if possible. We will update your Data Availability statement on your behalf to reflect the information you provide. Additional Editor Comments: Dear Authors, Please address the following points raised by the reviewer. The mesenchymal stem cell-conditioned medium gives promising results in in vitro and in vivo studies, and it is exciting to see that these mediums can be applied to humans. Therefore, the presented work is valuable, but some requirements must be met in order to be publishable. Introduction 1. The introduction is weak for this study. The importance of using the conditioned medium derived from mesenchymal stem cells for inflammatory diseases should be emphasized. The literature contains many in vitro and in vivo studies on this subject. The introduction should be supported by these studies. Why is it desired to be used in humans? This context should be well established. Methods 2. How many donors were used as sources of mesenchymal stem cells? Was CM prepared separately from the cells obtained from these donors? Which source was the CM used for which disease? It should be specified. 3. Has the content of CM obtained from adipose tissue been compared with that of CM obtained from the umbilical cord? This difference is significant, and the study should have been planned with this in mind. 4. The preparation conditions of CMs should be explained in the method section, such as the number of cells, how long they were kept, how much medium, medium, and supplement content were used, etc. 5. How were the doses determined? Your sample size for INH is 1. This is insufficient. Results 6. What do 1 and 2 represent in Figure 2? The x-axis naming of all graphs should be corrected. Label in an understandable way when looking at the graph or add a description to the figure legend. It is irrational for the "error bar SE" label to be on the x-axis. Add this to the figure legend and label it in a way that makes your data understandable. Discussion 7. The findings should be supported by literature data in the discussion. Possible safety and side effects should be discussed with possible reasons, considering variables such as administration routes, CM source used, disease, etc. Thanks Shaifur Rahman, PhD [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 ******** 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 Reviewer #1: No ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes ****** Reviewer #1: The mesenchymal stem cell-conditioned medium gives promising results in in vitro and in vivo studies, and it is exciting to see that these mediums can be applied to humans. Therefore, the presented work is valuable, but some requirements must be met in order to be publishable. Introduction 1. The introduction is weak for this study. The importance of using the conditioned medium derived from mesenchymal stem cells for inflammatory diseases should be emphasized. The literature contains many in vitro and in vivo studies on this subject. The introduction should be supported by these studies. Why is it desired to be used in humans? This context should be well established. Methods 2. How many donors were used as sources of mesenchymal stem cells? Was CM prepared separately from the cells obtained from these donors? Which source was the CM used for which disease? It should be specified. 3. Has the content of CM obtained from adipose tissue been compared with that of CM obtained from the umbilical cord? This difference is significant, and the study should have been planned with this in mind. 4. The preparation conditions of CMs should be explained in the method section, such as the number of cells, how long they were kept, how much medium, medium, and supplement content were used, etc. 5. How were the doses determined? Your sample size for INH is 1. This is insufficient. Results 6. What do 1 and 2 represent in Figure 2? The x-axis naming of all graphs should be corrected. Label in an understandable way when looking at the graph or add a description to the figure legend. It is irrational for the "error bar SE" label to be on the x-axis. Add this to the figure legend and label it in a way that makes your data understandable. Discussion 7. The findings should be supported by literature data in the discussion. Possible safety and side effects should be discussed with possible reasons, considering variables such as administration routes, CM source used, disease, etc. ****** 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.] 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. Attachments Attachment Submitted filename: Review report-12.08.2024.docx https://doi.org/10.1371/journal.pone.0322497.r002
Revision 1
1 Nov 2024 Author Response Response to Reviewers 1. The introduction is weak for this study. The importance of using the conditioned medium derived from mesenchymal stem cells for inflammatory diseases should be emphasized. The literature contains many in vitro and in vivo studies on this subject. The introduction should be supported by these studies. We have introduced new reviews and articles for heart failure and lung disease to the introduction. Many in vitro reports are cited in these studies, and treatment with adipose-derived stem cells has been shown to be effective. Several articles were also cited for kidney failure. These articles have shown that umbilical cord-derived mesenchymal stem cells (MSCs) are effective, and the main effect is thought to be the anti-inflammatory effect of factors released from the cells. Why is it desired to be used in humans? This context should be well established. We described that stem cell therapy is widely used, compared stem cell therapy with treatment using the conditioned medium, showed that treatment using the conditioned medium has many advantages from both the patient and manufacturing perspectives, and provided the justification for starting conditioned medium treatment in patients. Methods 2. How many donors were used as sources of mesenchymal stem cells? Was CM prepared separately from the cells obtained from these donors? We have included additional descriptions of the donors and CM preparation under the headings “Conditioned medium used for treatment,” and “Comparison on MSC-CMs. Which source was the CM used for which disease? It should be specified. The conditioned medium that was used for each disease was described in the methods under the heading, “dosing methods.” 3. Has the content of CM obtained from adipose tissue been compared with that of CM obtained from the umbilical cord? This difference is significant, and the study should have been planned with this in mind. We included in vitro experimental data to strengthen the justification for the type of conditioned medium selected. 4. The preparation conditions of CMs should be explained in the method section, such as the number of cells, how long they were kept, how much medium, medium, and supplement content were used, etc. Thank you for your comment. We have included additional descriptions in the methods to descript the preparation conditions. 5. How were the doses determined? Your sample size for INH is 1. This is insufficient. Results For heart failure and lung disease cases, administration was conducted into the pulmonary artery via catheter. For kidney failure, administration was conducted into the descending aorta near the renal artery. Given the direct administration to the diseased site, we administered a large amount of the conditioned medium over a long period of time with the hope of seeing a significant effect. Considering the burden imposed on patients, we set an administration time of 24 hours. In Dahbour et al., (2017), , they administered an average of 18 mL of conditioned medium into the spinal cavity. Considering that arterial administration has more diffusion than thecal administration, we administered the approximately doubled amount (30 mL). The organ causing the disease is unknown for fatigue and chronic pain treatment, so intravenous administration was conducted with the aim of systemic diffusion. Considering the economic burden on patients, the amount was reduced to 10 mL compared to arterial administration. Additionally, we observed the patient’s condition for rapid diffusion and safety by avoiding administration over a very short period of time, instead conducting administration over the course of three hours. The above content was added to the “Materials and Methods” section. Regarding the INH sample size, this report is a summary of what was conducted since they are the results of an observational study and did not involve patient allocation. We also included in the Discussion that the number of patients needs to be increased, and patients need to be observed in order to make comparisons by administration route. 6. What do 1 and 2 represent in Figure 2? The x-axis naming of all graphs should be corrected. Label in an understandable way when looking at the graph or add a description to the figure legend. It is irrational for the "error bar SE" label to be on the x-axis. Add this to the figure legend and label it in a way that makes your data understandable. The “1” and “2” in Fig. 2 were specified as “before administration” and “after administration”. The X-axis was clarified as the CRP value. An explanation of the error bars is also provided in the legend. Discussion 7. The findings should be supported by literature data in the discussion. Possible safety and side effects should be discussed with possible reasons, considering variables such as administration routes, CM source used, disease, etc. The basis for selecting the target disease from the results of the comparative experiment of AD-MSC-CM and UC-MSC-CM is shown together with the literature data. We judged that the drug could be safely administered with no observed side effects regardless of the administration route, CM source, or target disease, which we described in the Discussion. We only evaluated safety in this study, so the sample size was insufficient for comparing differences in effectiveness due to different administration routes for each disease. We have also added that future study is necessary for determining whether similar effects could be achieved using methods that place less of a burden on patients. https://doi.org/10.1371/journal.pone.0322497.r003
29 Jan 2025 Decision Letter - Gianpaolo Papaccio, Editor Dear Dr. Inami, 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 answers of the Authors to the reviewers are almost all largely insufficient. In particular, n+but not only, the answer: "We judged that the drug could be safely administered with no observed side effects regardless of the administration route, CM source, or target disease, which we described in the Discussion. We only evaluated safety in this study, so the sample size was insufficient for comparing differences in effectiveness due to different administration routes for each disease. We have also added that future study is necessary for determining whether similar effects could be achieved using methods that place less of a burden on patients" means that this study is extremely limited and does not merit publication. Please submit your revised manuscript by Mar 15 2025 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 . 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, Gianpaolo Papaccio, M.D., Ph.D. Academic Editor PLOS ONE Additional Editor Comments: The answers of the Authors to the reviewers are almost all largely insufficient. In particular, n+but not only, the answer: "We judged that the drug could be safely administered with no observed side effects regardless of the administration route, CM source, or target disease, which we described in the Discussion. We only evaluated safety in this study, so the sample size was insufficient for comparing differences in effectiveness due to different administration routes for each disease. We have also added that future study is necessary for determining whether similar effects could be achieved using methods that place less of a burden on patients" means that this study is extremely limited and does not merit publication. In addition the pitfalls contained in it as outlined by one reviewer lead to the thought that this is the last possibility they have to effectively amend the paper. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #1: (No Response) Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> Reviewer #1: Partly 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 Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: No Reviewer #2: Yes ****** Reviewer #1: 1. Overall, the new articles have improved the focus on the objective in the introduction. However, they should be presented in a more comparative and cohesive way, not just listed as separate studies. Improving the flow and removing repetitive content would make the section clearer and more concise. 2. The headings presented under the material method are mixed. This should be presented in a logical flow. Ethics should be at the beginning, statistics should be at the end, the dosing schedule should be followed by dosing methods, etc. The titles under the dosing method section should be structured as subheadings. However, another distinct method appears later, disrupting the organization. The section should be properly arranged to ensure clarity and consistency. 3. Although the article has an interesting topic and method, it needs improvement in writing and presentation of data. Reviewer #2: (No Response) ****** 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.0322497.r004
Revision 2
20 Mar 2025 Author Response Response to Reviewer’s Comments Reviewer Comments to Author 1. Overall, the new articles have improved the focus on the objective in the introduction. However, they should be presented in a more comparative and cohesive way, not just listed as separate studies. Improving the flow and removing repetitive content would make the section clearer and more concise. Reply: Thank you for your valuable feedback. We have revised the introduction to present the referenced studies in a more comparative and cohesive manner, improving the overall flow and removing redundant content. The therapeutic effects of conditioned medium (CM) have now been organized by target disease, highlighting its advantages over stem cell therapy. Additionally, to maintain focus on inflammation, we excluded chronic pain patients who did not undergo blood collection and whose CRP levels could not be assessed. To enhance clarity, we, have also updated the manuscript title to: "Safety assessment of multiple systemic administration of human mesenchymal stem cell-conditioned medium for various chronic diseases." 2. The headings presented under the material method are mixed. This should be presented in a logical flow. Ethics should be at the beginning, statistics should be at the end, the dosing schedule should be followed by dosing methods, etc. The titles under the dosing method section should be structured as subheadings. However, another distinct method appears later, disrupting the organization. The section should be properly arranged to ensure clarity and consistency. Reply: Thank you for your insightful comments. We have reorganized the Methods section for improved clarity and logical flow. Ethics is now positioned at the beginning, followed by the production methods and properties of CM. The administration schedule now precedes the administration methods, with titles properly structured as subheadings. Statistics have been placed at the end, and the previously disjointed section has been integrated for better coherence. 3. Although the article has an interesting topic and method, it needs improvement in writing and presentation of date. Reply: Thank you for your feedback. We have revised the manuscript to enhance clarity and ensure effective data presentation. Specifically, we have emphasized that safety was evaluated based on the absence of CRP level increases, indicating no progression of inflammation. https://doi.org/10.1371/journal.pone.0322497.r005
23 Mar 2025 Decision Letter - Gianpaolo Papaccio, Editor Safety assessment of multiple systemic administration of human mesenchymal stem cell-conditioned medium for various chronic diseases PONE-D-24-23481R2 Dear Dr. Inami, 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, Gianpaolo Papaccio, M.D., Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0322497.r006
Formally Accepted
Acceptance Letter - Gianpaolo Papaccio, Editor PONE-D-24-23481R2 PLOS ONE Dear Dr. Inami, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 customercare@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. Gianpaolo Papaccio Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0322497.r007

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .