Efficient immortalization of human dental pulp stem cells with expression of cell cycle regulators with the intact chromosomal condition

Ai Orimoto; Seiko Kyakumoto; Takahiro Eitsuka; Kiyotaka Nakagawa; Tohru Kiyono; Tomokazu Fukuda

doi:10.1371/journal.pone.0229996

Peer Review History

Original SubmissionDecember 16, 2019
7 Jan 2020 Decision Letter - Gianpaolo Papaccio, Editor PONE-D-19-34813 Efficient immortalization of human dental pulp stem cells with expression of cell cycle regulators with intact chromosomal condition PLOS ONE Dear Orimoto, 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. This study is of potential interest. but preliminary. As the reviewers specified the Authors must prove that cells do not die for senescence and keep their differentiating properties. To do so they must prove that after several passage they express antigens and are alive as well as that they are still capable to differentiate into osteogenic and adipogenic lineages. We would appreciate receiving your revised manuscript by Feb 21 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. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Gianpaolo Papaccio, M.D., Ph.D. 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. 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Please ensure that you refer to Figure 5 in your text as, if accepted, production will need this reference to link the reader to the figure. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: No Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: I Don't Know ****** 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: In this paper authors established a novel immortalized dental pulp stem cell line by co-expressing a mutant cyclin-dependent kinase 4 (CDK4 R24C ), Cyclin D1, and telomerase reverse transcriptase (TERT). They concluded that the established K4DT cell line has the advantage of being easy to handle, resulting useful in dental pulp regeneration therapy. The concept is interesting, but it represents only a preliminary study. The authors should test the characteristic of the new generated cell line, such as the stemness and the ability to differentiate at least in osteogenic lineage. For these reasons, the conclusions are not supported by data. Reviewer #2: In this manuscript the Authors describe a new immortalized cell line obtained from dental pulp stem cells infected with retrovirus carrying the genes for a mutant cyclin-dependent kinase 4 (CDK4R24C), Cyclin D1, and telomerase reverse transcriptase (TERT). The Authors showed that such cell line have a normal karyotype and do not go in senescence after few passages. The Authors propose this cell line as a new tool to study dental pulp stem cells based regeneration protocols. The work is interesting and the experiments are clearly performed, however there are few concerns that need to be addressed a s follow: In order to be used as a tool for regeneration experiments it not sufficient to prove that the cells do not go in senescence, the author should also prove that the cells keep their differentiation properties, so they should allow the cells to grow for few passages and then show that they are still able to differentiate toward at least osteogenic and adipogenic phenotype (just as a proof of concept). Dental pulp stem cells have been described to express different markers (see as an example “Methods for the identification, characterization and banking of human DPSCs: current strategies and perspectives. Stem Cell Rev Rep. 2011 Sep;7(3):608-15. doi: 10.1007/s12015-011-9235-9.) the Author should show that the expression of stemness associated markers is not lost after passages. ****** 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 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.0229996.r001
Revision 1
10 Feb 2020 Author Response Revision summary We would like to submit our revised version of our manuscript, which entitled “Efficient immortalization of human dental pulp stem cells with expression of cell cycle regulators with the intact chromosomal condition”. All comments obtained from the reviewers were quite productive to improve our manuscript. We will list all comments and corresponding change listed below with point by point style. Furthermore, we will submit our revised manuscript with changes highlighted. In this round of revisions, we performed flow cytometric analysis and the differentiation experiments to test the characteristic of our generated cell line. We showed that our established immortalized human dental pulp stem cells preserved the expression of stemness associated markers and their differentiation properties even after passages. As we carried out our best efforts to satisfy the comments, we are glad to finish up the additional task, if it is essential for the acceptance of the manuscript. 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 http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf We performed double-check the formatting style of the PLoS One, and re-formatted the entire manuscript. Now, we hope that our modification of the manuscript style would be satisfactory to the comments. 2. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability. Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized. Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. Based on the instruction, we included all experimental data used to the conclusions drawn in our manuscript. The data upload would be satisfactory to the comments from the journal office. 3. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. Based on the instruction, we uploaded full range blots and gel images as Supplementary Figures. 4. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ Based on the instruction, the corresponding author create a new ORCID iD. 5. Please ensure that you refer to Figure 5 in your text as, if accepted, production will need this reference to link the reader to the figure. Based on the instruction, we included Figure 5 in our manuscript to allow the reader to link the figure. Thank you for the suggestion. 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: In this paper authors established a novel immortalized dental pulp stem cell line by co-expressing a mutant cyclin-dependent kinase 4 (CDK4 R24C ), Cyclin D1, and telomerase reverse transcriptase (TERT). They concluded that the established K4DT cell line has the advantage of being easy to handle, resulting useful in dental pulp regeneration therapy. The concept is interesting, but it represents only a preliminary study. The authors should test the characteristic of the new generated cell line, such as the stemness and the ability to differentiate at least in osteogenic lineage. For these reasons, the conclusions are not supported by data. We appreciate very much to summarize the outline of our publication. Based on the comments, to test the characteristic of our generated cell line, termed K4DT cells, we performed flow cytometric analysis and the differentiation experiments. As a result, flow cytometry analysis of K4DT cells showed the high expression of the common stem cell markers in mesenchymal stem cell, CD90, and low expression of hematopoietic markers, CD34 and CD45, similar to the results observed in wild type cells. In the differentiation experiments, we tested the ability to differentiate in the osteogenic and adipogenic lineage. The K4DT cells were differentiated into osteoblasts immunostained with anti-osteocalcin antibody and showed marked mineralization by Alizarin Red S staining. K4DT cells also differentiated adipocytes and showed positive staining of numerous oil droplets by Oil red O. Our data suggest that our established cell line maintained its original stemness characteristics without affecting differentiation potential. To report the results described above, we involved new figures (Figure 7, 8, and 9 in the revised manuscript) and corresponding descriptions in the section of Materials and Methods (lines 157-183), Results (lines 248-268), and Discussion (lines 323-336). Reviewer #2: In this manuscript the Authors describe a new immortalized cell line obtained from dental pulp stem cells infected with retrovirus carrying the genes for a mutant cyclin-dependent kinase 4 (CDK4R24C), Cyclin D1, and telomerase reverse transcriptase (TERT). The Authors showed that such cell line have a normal karyotype and do not go in senescence after few passages. The Authors propose this cell line as a new tool to study dental pulp stem cells based regeneration protocols. The work is interesting and the experiments are clearly performed, however there are few concerns that need to be addressed a s follow: In order to be used as a tool for regeneration experiments it not sufficient to prove that the cells do not go in senescence, the author should also prove that the cells keep their differentiation properties, so they should allow the cells to grow for few passages and then show that they are still able to differentiate toward at least osteogenic and adipogenic phenotype (just as a proof of concept). Dental pulp stem cells have been described to express different markers (see as an example “Methods for the identification, characterization and banking of human DPSCs: current strategies and perspectives. Stem Cell Rev Rep. 2011 Sep;7(3):608-15. doi: 10.1007/s12015-011-9235-9.) the Author should show that the expression of stemness associated markers is not lost after passages. We appreciate very much to summarize the outline of our publication and for the critical and productive comments to our publication. Based on the suggestion, to determine whether our immortalized cells, termed K4DT cells, keep the original characteristic of dental pulp stem cells, the stemness and their differentiation abilities were assessed by using the K4DT cells at passage 8, 15, or 16. As described in the responses to the Reviewer’s comment #1, flow cytometry analysis of K4DT cells at passage 8 showed the high expression of the common stem cell markers in mesenchymal stem cell, CD90 and low expression of hematopoietic markers, CD34 and CD45, similar to the results observed in wild type cells. In addition, we studied their differentiation abilities for osteogenesis and adipogenesis by using the K4DT cells at passage15 and 16. As a result, we observed that the K4DT cells differentiated into osteoblasts and adipocytes in each differentiation medium containing differentiation inducers. K4DT cells at passages 15 after approximately 3 months that started cell culture showed positive staining of calcified materials by Alizarin red S after 17 days. The K4DT cells also differentiated into osteoblasts immunostained with anti-osteocalcin antibody after 25 days. After 23 days of culture with adipoinduction medium, the K4DT at passages 16 developed numerous lipid droplets stained with Oil Red O. These results indicated that our established immortalized human dental pulp stem cells preserved the expression of stemness associated markers and their differentiation properties even after passages. To report the results described above, we involved new figures (Figure 7, 8, and 9 in the revised manuscript) and corresponding descriptions in the section of Materials and Methods (lines 157-183), Results (lines 248-268), and Discussion (lines 323-336). Attachments Attachment Submitted filename: Revision summary plos 021020 3_.doc https://doi.org/10.1371/journal.pone.0229996.r002
19 Feb 2020 Decision Letter - Gianpaolo Papaccio, Editor PONE-D-19-34813R1 Efficient immortalization of human dental pulp stem cells with expression of cell cycle regulators with the intact chromosomal condition PLOS ONE Dear Orimoto, 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 paper has been improved but requires some minor amendments as specified below. ============================== We would appreciate receiving your revised manuscript by Apr 04 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. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Gianpaolo Papaccio, M.D., Ph.D. Academic Editor PLOS ONE Additional Editor Comments (if provided): The manuscript is partially improved. Actually the Authors must add either in the Introduction and in the Discussion Sections some paragraphs regarding the osteogenic differentiation that DPSCs spontaneously undergo, citing the following previous literature: Clin Sci. 131, Issue 8, 2017, Pages 699-713 regarding the capability of DPSCs to build a human bone tissue as well as J cell Physiol 2013, 228, pp 1149/53 regarding osteocalcin. Moreover they must add some info regarding the methods previously raised citing Stem Cell Rev rep 2011, 7: 608/15. [Note: HTML markup is below. Please do not edit.] 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: (No Response) Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: (No Response) 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 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: (No Response) Reviewer #2: Yes ****** 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: (No Response) 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: (No Response) Reviewer #2: The Authors have adressed the comments raised by this reviewer. The manuscriptresults greatly improved. ****** 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 [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.0229996.r003
Revision 2
19 Feb 2020 Author Response February 20, 2020 Editorial Office PLOS ONE Dear Professor Gianpaolo Papaccio We have attached our revised manuscript (PONE-D-19-34813R1) file entitled ‘: Efficient immortalization of human dental pulp stem cells with expression of cell cycle regulators with the intact chromosomal condition.’ We appreciate your invitation to submit a revised manuscript and have provided an improved manuscript. We also provide responses for Additional Editor Comments. Furthermore, we will submit our revised manuscript with changes highlighted. As we carried out our best efforts to satisfy the comments, we are happy to finish up the additional task, if it is essential for the acceptance of the manuscript. Additional Editor Comments (if provided): The manuscript is partially improved. Actually the Authors must add either in the Introduction and in the Discussion Sections some paragraphs regarding the osteogenic differentiation that DPSCs spontaneously undergo, citing the following previous literature: Clin Sci. 131, Issue 8, 2017, Pages 699-713 regarding the capability of DPSCs to build a human bone tissue as well as J cell Physiol 2013, 228, pp 1149/53 regarding osteocalcin. We appreciate very much for the supportive comments to our publication. We agree with this comment that we must add these references. As suggested, we have added two references as suggested, and have improved the corresponding manuscript at line 49-51 and lines 261-262. Moreover they must add some info regarding the methods previously raised citing Stem Cell Rev rep 2011, 7: 608/15. Thank you for your suggestion. As suggested, we included the reference in the section of Discussion and improved the corresponding manuscript at line 326-329. We hope that the revised manuscript is now acceptable for publication in Plos one. If you require any further information, please do not hesitate to contact me. Sincerely, Ai Orimoto Ai Orimoto, DDS, Ph. D. Graduate School of Science and Engineering, Iwate University, 4-3-5, Ueda, Morioka, Iwate, 020-8551, Japan TEL: 81-19-621-6375 E-mail: orimoto@ iwate-u.ac.jp https://doi.org/10.1371/journal.pone.0229996.r004
20 Feb 2020 Decision Letter - Gianpaolo Papaccio, Editor Efficient immortalization of human dental pulp stem cells with expression of cell cycle regulators with the intact chromosomal condition PONE-D-19-34813R2 Dear Dr. Orimoto, 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, Gianpaolo Papaccio, M.D., Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): The Authors answered to all the previous comments. Reviewers' comments: https://doi.org/10.1371/journal.pone.0229996.r005
Formally Accepted
24 Feb 2020 Acceptance Letter - Gianpaolo Papaccio, Editor PONE-D-19-34813R2 Efficient immortalization of human dental pulp stem cells with expression of cell cycle regulators with the intact chromosomal condition Dear Dr. Orimoto: I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Prof. Gianpaolo Papaccio Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0229996.r006

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