3D culture of functional human iPSC-derived hepatocytes using a core-shell microfiber

Shogo Nagata; Fumisato Ozawa; Minghao Nie; Shoji Takeuchi

doi:10.1371/journal.pone.0234441

Peer Review History

Original SubmissionFebruary 20, 2020
31 Mar 2020 Decision Letter - Hiroaki Onoe, Editor PONE-D-20-04950 3D culture of functional human iPSC-derived hepatocytes using a core-shell microfiber PLOS ONE Dear Prof. Takeuchi, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. We would appreciate receiving your revised manuscript by May 15 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. 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Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests [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: Yes Reviewer #2: Partly Reviewer #3: Yes Reviewer #4: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know Reviewer #2: Yes Reviewer #3: Yes Reviewer #4: 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 Reviewer #3: Yes Reviewer #4: 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 Reviewer #3: Yes Reviewer #4: 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 authors reported a novel cultivation method of Human iPSC-hepatocytes with a fiber-shaped 3D scaffold. They encapsulated hepatocytes into the core of core-shell hydrogel fibers to achieve both a 3D ECM-rich environment and easy manipulation. The approach is interesting and they showed that the protein secretion and gene expression increase compare to 2D or spheroid culture. However, the results are a bit unclear because the manuscript lacks some information or experiments as follows: 1. For line 185 and Fig. 3B, there is a contradiction in the culture time until evaluating the albumin secretion (The manuscript is written as day 3 & 7, while the figure is shown as day 3 & 6). They need to be corrected. 2. In Fig. 3C, 4A, 4B, and S4, the authors compared the gene expression of hepatocytes. However, there is no description of the normalization of cell numbers. Since the number of cells affects to the amount of expression, the results should be normalized for comparison. The description should be added. If the results are not the normalized one, they should be revised. 3. In Fig. 5C and lines 276-280, the authors indicated that the concentration of albumin increases by transplantation of fibers compared to the non-transplanted case. However, since the purpose of the authors is to increase the cell function by their culture method, the effect should be compared not with the non-transplanted case but with the previous culture methods (e.g. spheroids). Reviewer #2: Takeuchi and co-workers reported a culture system for human iPSC-derived hepatocyte-like cells using the core-shell microfibers. The authors compared the cell functions between the fiber and conventional 2D culture conditions, and claimed that the presented approach was effective. In addition, the authors perform in vivo experiments to show the applicability of the fiber to transplantation therapy. In general, organization of individual cells into 3D platforms is a good strategy to maximize the cell functions, and the presented paper is interesting as one of new approaches. However, following points should be properly explained and/or reflected before publication of this paper in this journal. (1) It was unclear why the authors obtained good results when they encapsulated cells in the fiber, because there is no rational explanation for this point. There should be some important cell-cell and cell-matrix interactions in 3D formats that potentially enhanced cell functions, with proper signal transductions. These points, especially from the viewpoint of the molecular signalings, should be properly explained. (2) The results of the animal study were not so convincing, because the authors did not mention the effectiveness of the fiber-based cell encapsulation and transplantation. Many researchers have transplanted cells (hepatocytes) that were encapsulated in hydrogel matrices, but the comparison with conventional strategies was not described at all in this paper. The reason for choosing the abdominal cavity as the transplantation site is also unclear; the liver itself, the kidney capsule, or subcutaneous site would be superior because of the presence of the blood flow. The blood concentration of human albumin might not be sufficiently high considering the number of the transplanted cells; I am wondering the cell viability. It was unclear what types of liver diseases could be improved by this approach, especially for humans. These points should be clarified. (3) It was unclear why the authors used Matrigel as the matrix, instead of collagen, the gold-standard for hepatocyte culture. (4) Some of the experimental conditions were not properly described. For example, following points are unclear: (i) the housekeeping gene used for qPCR, (ii) the age of the animal, and (iii) how many times the authors repeated the experiments, especially for the figures. (5) The manuscript contains many typos and unnatural expressions. The entire manuscript should be thoroughly checked once again before submitting the revision. Reviewer #3: This manuscript presents the 3D culture technique of human iPSC-derived hepatocytes using a core-shell microfiber. The core-shell microfiber is a unique, superior 3D culture platform that provides an ECM-abundant core and a mechanically-strong shell. The authors proved the potential of their original core-shell fibers for cell transplantation applications. The manuscript would seem of considerable interest to those working in tissue engineering and regenerative medicines. However, the authors should describe the differences with their previous publication in IEEE MEMS 2020 titled “3D hepatic tissue formed by iPSC-derived hepatocytes using a cell fiber technology.” Figure 1 includes the exactly same figures in Figure 3 of the previous publication. After polished based on the aforementioned critiques, this manuscript may be able to be published in PLOS ONE. I would recommend that this paper needs minor revision to be published in PLOS ONE. Reviewer #4: Nagata et al. apply cell fiber biofabrication techniques to create core-shell fibers of human iPSC-derived hepatocytes and perform in vitro and in vivo characterizations, including transplantation in a mouse model. Overall, the work is thorough; however, there are several aspects that should be expanded before publication. First, the introduction is missing a lot of prior work on cell fibers. The state of the art should be discussed in more detail (e.g., which cells have been demonstrated as compatible with the cell fiber approach, why haven't human iPSC-derived hepatocytes been done before, etc.). Such discussion on how this work is different from prior developments is important. I think a figure and supplementary movie featuring the biofabrication process is needed. Fabrication results are included in Fig. 2, but that figure should be expanded with the fabrication setup and process, which will be helpful for the readership. Also, the conclusion reads like a quick summary, but conclusions should really be used to provide some deeper insights into the study. There are some other minor notes, like the format of randomly including the figure captions in the main text being difficult for the reviewers and some weird callouts to the suppl. figures (S2A Fig). Overall though, should the aforementioned changes be made, my recommendation is for the manuscript to be accepted. ****** 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 Reviewer #3: No Reviewer #4: 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.0234441.r001
Revision 1
5 May 2020 Author Response Response to Reviewer# 1 Comment 1: For line 185 and Fig. 3B, there is a contradiction in the culture time until evaluating the albumin secretion (The manuscript is written as day 3 & 7, while the figure is shown as day 3 & 6). They need to be corrected. Response 1: We apologize for the incorrect information and have corrected the statement in the manuscript. Comment 2: In Fig. 3C, 4A, 4B, and S4, the authors compared the gene expression of hepatocytes. However, there is no description of the normalization of cell numbers. Since the number of cells affects to the amount of expression, the results should be normalized for comparison. The description should be added. If the results are not the normalized one, they should be revised. Response 2: We apologize for causing this misunderstanding. For analyzing gene expression, we used RT-PCR technique. In the assay, the gene expression level of each sample can be normalized by the expression level of the housekeeping gene instead of cell number. Here we choose GAPDH, which is widely used as the housekeeping gene, for the normalization, so it is not necessary to mention cell number of the samples. We have revised the sentence on quantitative RT-PCR in the materials and methods section. Comment 3: In Fig. 5C and lines 276-280, the authors indicated that the concentration of albumin increases by transplantation of fibers compared to the non-transplanted case. However, since the purpose of the authors is to increase the cell function by their culture method, the effect should be compared not with the non-transplanted case but with the previous culture methods (e.g. spheroids). Response 3: We appreciate the reviewer for this comment. In this study, we demonstrate that the cell fiber technique improve hepatocyte functionality in vitro, and we have not compared the function of the fibers as a transplant with conventional hepatocyte transplantation methods in the animal experiment. Here, we focus on showing the possibility of applying the hepatocyte fibers to cell transplantation therapy. This is because the transplantation of the hepatocyte fibers into the abdominal cavity performed in this study is a completely novel transplantation method, and thus it is difficult to compare with the conventional transplantation methods, in which hepatocytes are transplanted into the liver by vein administration of hepatocytes. Response to Reviewer# 2 Comment 1: It was unclear why the authors obtained good results when they encapsulated cells in the fiber, because there is no rational explanation for this point. There should be some important cell-cell and cell-matrix interactions in 3D formats that potentially enhanced cell functions, with proper signal transductions. These points, especially from the viewpoint of the molecular signalings, should be properly explained. Response 1: We appreciate the reviewer for this comment. We are also focusing on the molecular mechanism on the enhancement of hepatocyte characteristics of the encapsulated iPSC-hepatocytes in the fiber. This is still challenging and many researchers are investigating the issue. Here we evaluated global gene expression profiles and compared the whole-transcriptome between the hepatocytes in the fiber and them in the 2D culture condition using RNA-sequencing technique. We have found some genes as candidates for enhancing hepatocyte characteristics in the fiber, but the details of their molecular functions are still under analysis and further research is needed in the future. In this revision, we do not mention the molecular mechanism but show the data of the RNA-sequencing analysis in S4 Fig. Furthermore, we have revised the sentences as shown below. [Revised manuscript] (Results and Discussion section “To precisely investigate…” line 263) To precisely investigate effects of the encapsulation of the iPSC-hepatocytes into the fiber, RNA-sequencing was performed, and the 2D and the cell fiber culture condition was compared on global gene expression. In S4A Fig, the heat map of the one-way hierarchical clustering (1,053 genes satisfying with fold change 2 (FC2)), and top 30 genes which were upregulated or downregulated in the cell fiber were shown based on DEG analysis, indicating that global gene expression of the iPSC-hepatocytes was changed in the cell fiber. In addition, gene ontology analysis indicated that not only hepatocyte-specific pathways but also wide biological processes were changed (S4B Fig). Comment 2: The results of the animal study were not so convincing, because the authors did not mention the effectiveness of the fiber-based cell encapsulation and transplantation. Many researchers have transplanted cells (hepatocytes) that were encapsulated in hydrogel matrices, but the comparison with conventional strategies was not described at all in this paper. The reason for choosing the abdominal cavity as the transplantation site is also unclear; the liver itself, the kidney capsule, or subcutaneous site would be superior because of the presence of the blood flow. The blood concentration of human albumin might not be sufficiently high considering the number of the transplanted cells; I am wondering the cell viability. It was unclear what types of liver diseases could be improved by this approach, especially for humans. These points should be clarified. Response 2: We appreciate the reviewer for this comment. As the reviewer pointed out, there are reports on transplantation of hepatocytes encapsulated in the hydrogel, but, in most of the studies, hydrogel microbeads are used for the cell encapsulation as we mentioned in the manuscript. The advantage of our cell fiber technology compared to the microbeads is that, by using a hydrogel microfiber having core-shall structure, the core retained the encapsulated hepatocytes in the ECM-rich 3D microenvironment, while the outer shell composed of alginate hydrogel supported the physical strength and stability in vivo of the cell fiber. Furthermore, since the fiber is possible to be handled as a single transplant due to its shape and strength, the transplant operation can be simple. In addition, when a malignant event occurs in the transplant or when it is no longer needed, the transplanted fiber can be more efficiently retrieved from the host than the microbeads. In this study, we chose intraperitoneal transplantation of the fiber because it is generally known that transplantation of hydrogel encapsulated hepatocytes into the peritoneal cavity is an attractive option for the management of acute liver failure providing short-term support to allow native liver regeneration [PLoS One. 2014 Dec 1;9(12):e113609.]. As the reviewer commented, transplantation into regions rich in blood vessels (e.g. subcutaneous site) is especially required for organ transplantation that requires permanent expression of cellular function, but, in this study, hepatocyte fiber transplantation is thought as one of novel cell therapy techniques and required to express the function in the short term as mentioned above. To clearly show that, we have revised the sentence as shown below. [Revised manuscript] (Results and Discussion section “For transplantation,…” line 300) For transplantation, our cell fibers have two advantages over hydrogel microbeads, which are widely used for cell encapsulation and transplantation research: first, the fiber shape is continuous in comparison to the separated droplets/beads, and therefore, the fibers can be easily picked up by tweezers and be efficiently implanted and retrieved as a whole; second, the fiber shell consists of alginate hydrogel, which makes the fibers mechanically strong to prevent the unwanted broad dispersion of the transplanted cells away from the injection site (Fig 5A). Comment 3: It was unclear why the authors used Matrigel as the matrix, instead of collagen, the gold-standard for hepatocyte culture. Response 3: We appreciate the reviewer for this comment. Type I collagen has been known as a suitable ECM for hepatocytes, but Matrigel is also known to be one of the most useful matrices for static, traditional hepatocyte culture [Tissue Eng Part A. 2010 Mar; 16(3): 1075–1082.]. Here we chose Matrigel for constructing the cell fiber due to basal membrane components for supporting the iPSC-hepatocytes in the core region of the fiber. Since various gels can be used for the cell fiber formation, it is possible to use an arbitrary gel (e.g. collagen) depending on the application. Comment 4: Some of the experimental conditions were not properly described. For example, following points are unclear: (i) the housekeeping gene used for qPCR, (ii) the age of the animal, and (iii) how many times the authors repeated the experiments, especially for the figures. Response 4: We appreciate the reviewer for this comment and have revised the sentence in the materials and methods section. Comment 5: The manuscript contains many typos and unnatural expressions. The entire manuscript should be thoroughly checked once again before submitting the revision. Response 5: We have checked again and revised the manuscript according to advice from experts on English. Response to Reviewer# 3 Comment :This manuscript presents the 3D culture technique of human iPSC-derived hepatocytes using a core-shell microfiber. The core-shell microfiber is a unique, superior 3D culture platform that provides an ECM-abundant core and a mechanically-strong shell. The authors proved the potential of their original core-shell fibers for cell transplantation applications. The manuscript would seem of considerable interest to those working in tissue engineering and regenerative medicines. However, the authors should describe the differences with their previous publication in IEEE MEMS 2020 titled “3D hepatic tissue formed by iPSC-derived hepatocytes using a cell fiber technology.” Figure 1 includes the exactly same figures in Figure 3 of the previous publication. After polished based on the aforementioned critiques, this manuscript may be able to be published in PLOS ONE. I would recommend that this paper needs minor revision to be published in PLOS ONE. Response : We appreciate the reviewer for this comment. As pointed out by the reviewer, a related but preliminary work was reported in the MEMS conference. Comparing to the conference proceedings, we have significantly polished the initial ideas based on the molecular biological insight which was gained by new experiments that precisely evaluated the hepatic function and the cell characteristics and showed that the cell fiber was useful for efficient 3D culture system of the iPSC-hepatocytes and cell therapy for liver failure treatment. On the comment for figure 1, we modified and revised parts of the figure. Response to Reviewer# 4 Comment : Nagata et al. apply cell fiber biofabrication techniques to create core-shell fibers of human iPSC-derived hepatocytes and perform in vitro and in vivo characterizations, including transplantation in a mouse model. Overall, the work is thorough; however, there are several aspects that should be expanded before publication. First, the introduction is missing a lot of prior work on cell fibers. The state of the art should be discussed in more detail (e.g., which cells have been demonstrated ascompatible with the cell fiber approach, why haven't human iPSC-derived hepatocytes been done before, etc.). Such discussion on how this work is different from prior developments is important. I think a figure and supplementary movie featuring the biofabrication process is needed. Fabrication results are included in Fig.2, but that figure should be expanded with the fabrication setup and process, which will be helpful for the readership. Also, the conclusion reads like a quick summary, but conclusions should really be used to provide some deeper insights into the study. There are some other minor notes, like the format of randomly including the figure captions in the main text being difficult for the reviewers and some weird callouts to the suppl. figures (S2A Fig). Overall though, should the aforementioned changes be made, my recommendation is for the manuscript to be accepted. Response : We appreciate the reviewer for this comment. We considered adding a video of the fabrication process, but due to the limited activity of our research facility caused by the spread of the coronavirus infection (COVID-19), it is difficult to do it within the revise period. However, the biofabrication technique used in this study was basically the same as in the previous report [Nat Mater. 2013 Jun;12(6):584-90.], so the details can be omitted in this manuscript. The modification in the fiber fabrication is that, for the transplantation experiment, barium ions was used as the crosslinker of alginate gel for the fiber strength and in vivo stability. To clearly show that, we have revised the sentence as shown below. [Revised manuscript] (Materials and methods section “The fibers generated in the …” line 91) The fibers generated in the device were collected in a tube filled with CaCl2 (for in vitro assay) or BaCl2 solution (for in vivo assay), and were incubated in the collection bath for 10 min. As the reviewer pointed out, there have been reports on the cell fibers. The cell fiber technology is thought to be applicable to all cell types in principle, but previous reports mainly focused on the fabrication technology using various cell lines, which are relatively stable in cultivation. The difference between them and this study is that here we developed the fibers using the iPSC-derived functional cells, which can be directly applied to medical applications (e.g. cell therapy). This is the first report of the fiber composed of iPSC-hepatocytes, which required to preciously evaluate the cell characteristics and transplantation experiments as performed in this study. The paper format follows the PloS ONE submission rules. Therefore, the figure captions are inserted in the main text immediately after the paragraph that cites the figure. We appreciate the comment on S2A Fig and have revised the sentence as shown below. [Revised manuscript] (Results and Discussion section “We compared the…” line 183) We compared the morphology of the iPSC-hepatocytes cultured for a week in the conventional 2D, spheroid, and the cell fiber culture conditions. In the 2D culture, the hepatocytes attached onto the Matrigel-coated plate, and formed 2D cell-cell interactions and exhibited a cobblestone morphology, which is characteristic of typical hepatocytes. In the spheroid culture, loose spheroid in distorted shapes was generated (S2A Fig). In contrast, in the cell fiber culture, the iPSC-hepatocytes formed compact cell aggregates with various 3D structures (Fig 2B). Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0234441.r002
27 May 2020 Decision Letter - Hiroaki Onoe, Editor 3D culture of functional human iPSC-derived hepatocytes using a core-shell microfiber PONE-D-20-04950R1 Dear Dr. Takeuchi, 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, Hiroaki Onoe 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 Reviewer #3: All comments have been addressed Reviewer #4: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes Reviewer #4: (No Response) ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes Reviewer #4: (No Response) ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes Reviewer #4: (No Response) ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes Reviewer #4: (No Response) ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Authors have properly addressed my concerns in the revision, and I agree to their explanations. I have no more concerns on this article. Reviewer #2: The revised manuscript by Takeuchi and co-workers properly reflected most of my previous concerns, and now the paper was improved. This paper is a nice example of the hydrogel fiber-based cell culture technique, and hence, I recommned accepting this paper for publication. Reviewer #3: The authors replied to the reviewers' comments well and improved the manuscript. I recommend the manuscript for publication. Reviewer #4: (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 Reviewer #3: No Reviewer #4: No https://doi.org/10.1371/journal.pone.0234441.r003
Formally Accepted
2 Jun 2020 Acceptance Letter - Hiroaki Onoe, Editor PONE-D-20-04950R1 3D culture of functional human iPSC-derived hepatocytes using a core-shell microfiber Dear Dr. Takeuchi: 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 Dr. Hiroaki Onoe Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0234441.r004

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 .