Development of a method of passaging and freezing human iPS cell-derived hepatocytes to improve their functions

Jumpei Inui; Yukiko Ueyama-Toba; Seiji Mitani; Hiroyuki Mizuguchi

doi:10.1371/journal.pone.0285783

Peer Review History

Original SubmissionNovember 20, 2022
19 Dec 2022 Decision Letter - Hon Fai Chan, Editor PONE-D-22-31940Development of a method of passaging and freezing human iPS cell-derived hepatocytes to improve their functions.PLOS ONE Dear Dr. Mizuguchi, 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. Changes required for acceptance:1. Functional assays such as albumin ELISA and urea assay should be performed.2. Quantification of immunostaining results should be presented (e.g. Fig. 4C, D, E). Recommended changes:1. The quality of figures should be improved.2. Alternative cell dissociation methods should be explored.3. Other recommended changes as suggested by the reviewers. Please submit your revised manuscript by Feb 02 2023 11:59PM. 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Kind regards, Hon Fai Chan, PhD Academic Editor PLOS ONE Journal requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. [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: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The passaging and freezing of human iPS cell-derived hepatocytes is an important issue in this study. The authors describe methods for dissociating, cryopreserving and reseeding HLCs and conclude that hepatic function persists. However, the main problem with this paper is that the data lack quantification and controls to fully support the conclusions of the paper. 1) Fig4C lacks quantification and does not compare the ratio of ALB HNF4A CYP3A4 AAT before and after passaging. neither does Fig4D and E. 2) Fig4B suggests using ES as a negative control and Fresh PHH as a positive control. 3) The full text should be quantified using the ALB Secretion ELISA assay. 4) Fig6, Comparison of cell activity after recovery with different cryopreservation solutions. In addition to qPCR, more functional quantitative data should be used for comparison. Reviewer #2: The paper of Mizuguchi et al. describes an improved method for passaging and freezing hepatocyte-like cells derived from iPSC. The topic is interesting since PSC-derived models present many advantages such their availability and may offer a stable source of hepatocytes and could be maintained for longer periods, although the present protocols still present many limitations like immature phenotype or difficulties for passaging and cryopreserving them. The paper is well designed and written but it has limitations that make it not suitable for publication in its present. A detailed list if the points needing revision is provided below: 1. The quality of the figures should be deeply improved. The schemes used are so simplistic and could be also improved and make them more appealing. 2. The authors just analyzed the use of TriplE for passaging the cells although many authors have used other systems such as collagenase, dispase or cell dissociation buffer for cell passaging. The authors should explore the use of other methods that just require 5-10 minutes. 3. A more detailed transcriptomics analysis (at least 10 genes) would improve the quality of the manuscript. 4. Functionality of the cells in terms of albumin production and secretion and ureogenesis capability should be also included since these are specific hepatic functions that are normally analysed in HLC. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0285783.r001
Revision 1
18 Apr 2023 Author Response Dear Editor of PLoS ONE Thank you for your effort in reviewing our manuscript entitled “Development of a method of passaging and freezing human iPS cell-derived hepatocytes to improve their functions.” by J. Inui et al. We thoroughly revised the manuscript in keeping with editor’s and reviewers’ suggestions. The sentences we revised to the manuscript are shown in red. Point-by-point responses about editor’s and reviewers’ suggestions are given below. Changes required for acceptance: 1. Functional assays such as albumin ELISA and urea assay should be performed. Response: We compared albumin secretion, urea secretion, and CYP3A4 activity in HLCs after passage or cryopreservation in CB1 with those in HLCs before passage. The results showed that HLCs had these functions after passage or cryopreservation comparable to or better than those in HLCs before passage. Please see lines 216-221 and Fig.7. 2. Quantification of immunostaining results should be presented (e.g. Fig. 4C, D, E). Response: We found it difficult to quantify the immunofluorescent staining images. Therefore, we instead performed FACS analysis about the expression of the most important hepatocyte markers, ALB and CYP3A4. The results showed that the percentage of ALB- or CYP3A4-positive cells increased in the HLCs after passage compared to before passage. Please see lines 170–174 and Fig.S6. In the results in Fig. 4D and E, the analyses were performed to confirm that HLCs after passage still had the capacity for glycogen storage and LDL uptake, and did not indicate that these functions were higher than before passage. Recommended changes: 1. The quality of figures should be improved. Response: We corrected the data with increased resolution. 2. Alternative cell dissociation methods should be explored. Response: We showed the results using other dissociation enzymes [dispase & collagenase (DC), TrypLE select (TS) and accutase (Ac)] in Fig. S5, and decided to use TrypLE select, which showed a more favorable trend, although not significantly different. We tried to dissociate HLCs with 5 minutes treatment of TrypLE select, but the cells were not recovered efficiently enough to perform the experiments, as shown in Fig. 3B. 3. Other recommended changes as suggested by the reviewers. Response: We thoroughly revised the manuscript and figures in keeping with each reviewer's suggestions. Point-by-point responses about reviewers’ suggestions are given below. Reviewer #1: 1. Fig4C lacks quantification and does not compare the ratio of ALB HNF4A CYP3A4 AAT before and after passaging. neither does Fig4D and E. Response: 　As the reviewer suggested, we performed FACS analysis to determine the percentage of ALB- or CYP3A4-positive cells before and after passage. The results showed that the percentage of ALB- or CYP3A4-positive cells increased in the HLCs after passage compared to before passage. Please see lines 170–174 and Fig.S6. In the results in Fig. 4D and E, the analyses were performed to confirm that HLCs after passage still had the capacity for glycogen storage and LDL uptake, and did not indicate that these functions were higher than those before passage. 2. Fig4B suggests using ES as a negative control and Fresh PHH as a positive control. Response: Since HLCs are derived from human iPS cells, we measured CYP3A4 activity in human iPS cells as a negative control and found no activity (data not shown). Most pharmaceutical companies perform drug toxicity studies with PHHs cultured for 48 to 72 hours. Therefore, in this study, we used PHHs cultured for 48 hours as a control to demonstrate the usefulness of HLCs after passage for drug toxicity studies. 3. The full text should be quantified using the ALB Secretion ELISA assay. Response: 　As the reviewer suggested, we compared the levels of albumin secretion in HLCs after passage or cryopreservation with those in HLCs before passage. The results showed that the levels of albumin secretion increased significantly in HLCs after passage, and this trend was maintained even after cryopreservation. Please see lines 216-221 and Fig.7. 4. Fig6, Comparison of cell activity after recovery with different cryopreservation solutions. In addition to qPCR, more functional quantitative data should be used for comparison. Response: 　As the reviewer suggested, we compared albumin secretion, urea secretion, and CYP3A4 activity in HLCs after passage or cryopreservation in CB1 with those in HLCs before passage. The results showed that HLCs after cryopreservation had these functions comparable to or better than the HLCs before passage, although urea secretion and CYP3A4 activity in the HLCs after cryopreservation were lower than those in the HLCs after passage. Please see lines 216-221 and Fig.7. Reviewer #2: 1. The quality of the figures should be deeply improved. The schemes used are so simplistic and could be also improved and make them more appealing. Response: We corrected the data with increased resolution. 　 2. The authors just analyzed the use of TriplE for passaging the cells although many authors have used other systems such as collagenase, dispase or cell dissociation buffer for cell passaging. The authors should explore the use of other methods that just require 5-10 minutes. Response: We showed the results using other dissociation enzymes [dispase & collagenase (DC), TrypLE select (TS) and accutase (Ac)] in Fig. S5, and decided to use TrypLE select, which showed a more favorable trend, although not significantly different. We tried to dissociate HLCs with 5 minutes treatment of TrypLE select, but the cells were not recovered efficiently enough to perform the experiments, as shown in Fig. 3B. 3. A more detailed transcriptomics analysis (at least 10 genes) would improve the quality of the manuscript. Response: 　As the reviewer suggested, we additionally examined the gene expression levels of more than 10 hepatocyte markers. Most genes were highly expressed in HLCs after passage or cryopreservation compared to those in HLCs before passage. Please see lines 185-187, 213-215, and Fig.S7 & S8. 4. Functionality of the cells in terms of albumin production and secretion and ureogenesis capability should be also included since these are specific hepatic functions that are normally analysed in HLC. Response: 　As the reviewer suggested, we compared albumin secretion and urea secretion in HLCs after passage or cryopreservation in CB1 with those in HLCs before passage. The results showed that HLCs had these functions after passage or cryopreservation comparable to or better than those in HLCs before passage, although urea secretion in the HLCs after cryopreservation was lower than that in the HLCs after passage. Please see lines 216-221 and Fig.7. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0285783.r002
2 May 2023 Decision Letter - Hon Fai Chan, Editor Development of a method of passaging and freezing human iPS cell-derived hepatocytes to improve their functions. PONE-D-22-31940R1 Dear Dr. Mizuguchi, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Hon Fai Chan, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: 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 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: No ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors have supplied additional data according to the comments. The revised manuscript mostly meets the requirements. However, the quality of figures are still not good enough. It's difficult to identify the cell morphological differences. Reviewer #2: (No Response) ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0285783.r003
Formally Accepted
9 May 2023 Acceptance Letter - Hon Fai Chan, Editor PONE-D-22-31940R1 Development of a method of passaging and freezing human iPS cell-derived hepatocytes to improve their functions. Dear Dr. Mizuguchi: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Professor Hon Fai Chan Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0285783.r004

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