Human umbilical cord-derived mesenchymal stem cells alleviate autoimmune hepatitis by inhibiting hepatic ferroptosis

Yaqin Li; Bing Liu; Guoxin Hu; Tao Zhou; Xuanqiu He; Ling Guo; Luoshi Zhang; Weizhao Tong; Yihua Chen; Youhua Xu; Guangdong Tong; Wei V. Zheng

doi:10.1371/journal.pone.0337060

Peer Review History

Original SubmissionJune 24, 2025
23 Jul 2025 Decision Letter - Junzheng Yang, Editor Dear Dr. Zheng, 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. Please submit your revised manuscript by Sep 06 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'. 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[Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? Reviewer #1: Yes Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: No Reviewer #2: N/A ****** 3. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: Page14/line5: `randomly assigned into four groups (n = 3 per group)`, the number in each group is too small and impossible to run the statistics. Please add more animals in each group and reevaluate the experiment. Page14/line8-9: how do you determine the cell number to inject. 1x10^6 cells per animal is too big amount. Is there any scientific reasons or references to support choosing this number. Please provide BW and gender in each group. After cell injection, how many hUC-MSCs were reached and retained in the liver. Stastics: "GraphPad Prism8" is application name, please provide what kind of statsitic was used in each experiment. Figue2: since upper and lower panel did not match, it is confusing to understand. In panel B, what yellow dots and white dots are indicating. Reviewer #2: the manuscript presents a comprehensive and well-structured investigation into the therapeutic potential of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) in autoimmune hepatitis (AIH), particularly focusing on the suppression of ferroptosis as a key protective mechanism 1. Abstract Revision – Improve Specificity and Flow Current Issue: The abstract is informative but lacks precision in quantitative terms and slightly overstates mechanistic conclusions. 2. Sample Size Limitation, Only n = 3 per group in key experiments. While preliminary data is still valuable, such a small number limits statistical power and generalizability. A sample size of 3 severely underpowers the study and affects statistical reliability. 3. Mechanistic Causality Needs Clarification While ferroptosis marker modulation is demonstrated, causal inference remains correlative. so the paper lack of Causal Validation of Ferroptosis Pathway 4. Missing Controls: A ConA + vehicle control group (without MSCs) for comparison in qPCR and Western blot analysis should be explicitly stated and shown. 5. There is minimal detail on how hUC-MSCs were characterized for identity, purity, and viability before administration. 6. Clarify Timing of Sampling Post-ConA. Inconsistent Statements: “24h after ConA injection” is mentioned in methods, but survival data are observed up to 18h. ****** what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy Reviewer #1: No Reviewer #2: Yes: Ramada R. K. ******** [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.0337060.r001
Revision 1
4 Sep 2025 Author Response Reviewer #1: Page14/line5: `randomly assigned into four groups (n = 3 per group), the number in each group is too small and impossible to run the statistics. Please add more animals in each group and reevaluate the experiment. Response: We appreciate the reviewer’s concern. We repeated the experiments in an additional six mice and incorporated the new data into the revised figures. One important clarification regarding the repeated experiments is that the two GLU measurements were performed using different assay kits. The original data were obtained with the MEIMIAN Glutamic Acid (Glu) Assay Kit (Cat. No. ADS-F-AJS007), whereas the repeat experiments used the Elabscience Glu Colorimetric Assay Kit (Cat. No. E-BC-K903-M). Owing to differences in the calculation methodologies between the two kits, the absolute values varied. Importantly, however, the intergroup trends remained consistent across both experiments. Page14/line8-9: how do you determine the cell number to inject. 1x10^6 cells per animal is too big amount. Is there any scientific reasons or references to support choosing this number. Please provide BW and gender in each group. After cell injection, how many hUC-MSCs were reached and retained in the liver. Response: The injection of 106 MSCs is a commonly used dosage in similar studies. Numerous publications have employed this dosage (e.g., PMID: 22752635, PMID: 33757765, PMID: 31133062). In the revised manuscript, we have included the baseline body weight of the mice (approximately 22-25 grams per mouse) before the experiments. The gender of the animals (female) was clearly stated in the original manuscript. Regarding the amount of hUC-MSCs homing to the liver, we did not specifically quantify the exact cell number. However, we generated GFP-labeled hUC-MSCs and injected them into mice under different treatment conditions. As shown in the revised Figure S2, hUC-MSCs were able to reach and be retained in the liver, with higher levels observed in injured liver tissues. We believe these results provide sufficient evidence to support our conclusion. Stastics: "GraphPad Prism8" is application name, please provide what kind of statsitic was used in each experiment. Response: The methods for statistical analyses had been clearly described in the figure legends. Figue2: since upper and lower panel did not match, it is confusing to understand. In panel B, what yellow dots and white dots are indicating. Response: Thank you for your comment. In the revised Figure 2, we have added dashed boxes in parts A and B to clearly indicate the correspondence between the upper and lower panels. The lower panels present magnified views of the specific regions highlighted in the upper panels. Yellow dots mark TUNEL-positive cells. The white areas are likely due to exceptionally strong apoptotic fluorescence signals from individual cells. Reviewer #2: the manuscript presents a comprehensive and well-structured investigation into the therapeutic potential of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) in autoimmune hepatitis (AIH), particularly focusing on the suppression of ferroptosis as a key protective mechanism 1. Abstract Revision – Improve Specificity and Flow Current Issue: The abstract is informative but lacks precision in quantitative terms and slightly overstates mechanistic conclusions. Response: Thank you for your comment. We have revised the Abstract to enhance its conciseness and precision, ensuring that it now more accurately reflects the key findings and perspectives of our study. We appreciate your feedback, which has been invaluable in improving the clarity of our manuscript. 2. Sample Size Limitation, Only n = 3 per group in key experiments. While preliminary data is still valuable, such a small number limits statistical power and generalizability. A sample size of 3 severely underpowers the study and affects statistical reliability. Response: Thank you for your comment. We have repeated the experiments using six additional mice and incorporated the new results into the revised figures. 3. Mechanistic Causality Needs Clarification While ferroptosis marker modulation is demonstrated, causal inference remains correlative. so the paper lack of Causal Validation of Ferroptosis Pathway Response: We understand the reviewer’s concern and have acknowledged this limitation in the Discussion section. Indeed, the exploration of ferroptosis-related mechanisms in this study remains relatively preliminary. While our current work primarily demonstrates an association between MSC-based therapy and ferroptosis inhibition, a more in-depth investigation into the underlying mechanisms will be pursued in a separate project using a broader range of experimental approaches. To further address this issue in the present study, we plan to conduct additional rescue experiments, such as the application of ferroptosis agonists, to clarify the causal relationship between the protective effects of MSCs and ferroptosis inhibition. We will publish these data in the future. 4. Missing Controls: A ConA + vehicle control group (without MSCs) for comparison in qPCR and Western blot analysis should be explicitly stated and shown. Response: We suspended the MSCs in PBS and administered the same volume of PBS, used for MSC suspension, to the mice in the ConA group, serving as the appropriate control as suggested by the reviewer. This information has been included in the revised manuscript. 5. There is minimal detail on how hUC-MSCs were characterized for identity, purity, and viability before administration. Response: We apologize for the missing details and have included them in the revised manuscript and Figure S1. 6. Clarify Timing of Sampling Post-ConA. Inconsistent Statements: “24h after ConA injection” is mentioned in methods, but survival data are observed up to 18h. Response: The survival data (Figure A) show that all mice in the ConA group died within 18 hours, whereas all mice in the other groups survived for at least 24 hours, as indicated by the red line at the top. Attachments Attachment Submitted filename: response to reviewers comments.docx https://doi.org/10.1371/journal.pone.0337060.r002
10 Oct 2025 Decision Letter - Ramada Khasawneh, Editor Dear Dr. Zheng, 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. Please submit your revised manuscript by Nov 23 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, Ramada Rateb Khasawneh Academic Editor PLOS ONE Journal Requirements: 1. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. 3. Please provide the original blot images for Figure 2S. 4. Please clarify the reasons for the authorship changes and specify why the changes were not made at initial submission. Additional Editor Comments (if provided): It is a good paper overall, but there are a few technical points I have raised. The abstract mentions “metabolomic analysis revealed modulation of pathways related to ferroptosis,” but does not specify whether pathway enrichment or specific metabolites were validated. A clearer statement of the level of evidence (correlation vs. validation) would strengthen accuracy. The abstract implies that hUC-MSCs inhibit ferroptosis as the mechanistic basis of protection (“through inhibiting the ferroptosis pathway”). Unless ferroptosis inhibition was directly tested (e.g., by using ferroptosis activators/inhibitors or rescue assays), it would be more accurate to phrase this as: “...potentially through modulation of ferroptosis-related pathways.” “hUC-MSCs” defined twice — keep only once The ConA model indeed induces T-cell–mediated immune hepatitis, but calling it “autoimmune hepatitis” should be qualified — it is an AIH-like murine model, not true autoimmune hepatitis .. please correct this [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #1: All comments have been addressed 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??> Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: I Don't Know Reviewer #3: 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 Reviewer #3: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** Reviewer #1: Authors addressed reviwer1's concern adequately. Reviewer1 has no further question on this manuscript. Reviewer #2: The author has carefully and comprehensively addressed all the reviewers’ comments and concerns. All issues raised during the review process have been adequately resolved, and the revised manuscript now meets the journal’s standards in terms of scientific quality, clarity, and presentation. Therefore, I believe the paper is now suitable for publication in its current form. Reviewer #3: This manuscript is thorough and technically sound, and it is clear that the authors have addressed all previous reviewers' concerns. ****** 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 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.] 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.0337060.r003
Revision 2
11 Oct 2025 Author Response 1. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. 3. Please provide the original blot images for Figure 2S. Response: Thanks for the comment. We have provided the original blot image for panel C of Figure S2 in the separate file "uncropped western blot-2". Please note that this is the only panel from this figure S2 for which a new original blot is required. 4. Please clarify the reasons for the authorship changes and specify why the changes were not made at initial submission. Response: Thanks for the comment.During the revision process, we have added Mr. Luoshi Zhang as a co-author to the manuscript. His substantial contributions to performing the critical experiments outlined in the response letter and his analysis of the new data fully meet the criteria for authorship. Additional Editor Comments (if provided): It is a good paper overall, but there are a few technical points I have raised. The abstract mentions “metabolomic analysis revealed modulation of pathways related to ferroptosis,” but does not specify whether pathway enrichment or specific metabolites were validated. A clearer statement of the level of evidence (correlation vs. validation) would strengthen accuracy. Response: Thanks for the advice. We have rephrased the sentence as follows: “Metabolomic and ontology analyses of mouse liver tissue samples revealed that hUC-MSCs treatment altered the levels of metabolites (Glu derivatives and peptides) functionally associated with ferroptosis-related pathways.” Since we didn’t perform validation, emphasizing ontology analysis should be sufficient to clarify this. The abstract implies that hUC-MSCs inhibit ferroptosis as the mechanistic basis of protection (“through inhibiting the ferroptosis pathway”). Unless ferroptosis inhibition was directly tested (e.g., by using ferroptosis activators/inhibitors or rescue assays), it would be more accurate to phrase this as: “...potentially through modulation of ferroptosis-related pathways.” “hUC-MSCs” defined twice — keep only once Response: We appreciate the advice. The relevant text has been modified accordingly. Yet, we can’t find a duplicated definition of hUC-MSCs in the Abstract. Following your suggestion, we have removed the redundant definition of hUC-MSCs from the Introduction, while retaining the single definition in the Abstract. The ConA model indeed induces T-cell–mediated immune hepatitis, but calling it “autoimmune hepatitis” should be qualified — it is an AIH-like murine model, not true autoimmune hepatitis .. please correct this Response: Thanks for the advice. We have made the modification in the revised manuscript. As suggested, we have changed "ConA-induced AIH mouse model" to "ConA-induced AIH-like mouse model" throughout the manuscript. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0337060.r004
4 Nov 2025 Decision Letter - Ramada Khasawneh, Editor Human Umbilical Cord-Derived Mesenchymal Stem Cells Alleviate Autoimmune Hepatitis by Inhibiting Hepatic Ferroptosis PONE-D-25-34339R2 Dear Dr. Zheng, 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. For questions related to billing, please contact billing support . 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, Ramada Rateb Khasawneh Academic Editor PLOS ONE Additional Editor Comments (optional): nice paper .. Good Luck Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 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??> Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: I Don't Know ****** 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: Yes Reviewer #2: Yes ****** Reviewer #1: Authors addressed reviewer’s concerns.Reviewer has no further comment. I agree to publish this manuscirpt. Reviewer #2: The article appears to be in excellent condition following the recent revisions. The authors have effectively addressed the previous comments and substantially improved the clarity, organization, and overall scientific quality of the manuscript. In its current form, the paper seems well-prepared and suitable for publication. ****** 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.0337060.r005
Formally Accepted
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