Serine Metabolism Regulates the Replicative Senescence of Human Dental Pulp Cells through Histone Methylation

Response to Reviewer 1 Comments

1. Summary

I would like to express our sincere appreciations of your constructive comments concerning our article entitled “Serine Metabolism Regulates the Replicative Senescence of Human Dental Pulp Cells through Histone Methylation”（ID：cimb-2897366）. These comments are all valuable and helpful for improving our article. According to your comments, we have made extensive modifications to our manuscript. Revised portions are marked in red in the manuscript.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

We have provided our response in the point-by-point response letter. The same as below.

Are all the cited references relevant to the research?

Yes

Is the research design appropriate?

Can be improved

Are the methods adequately described?

Can be improved

Are the results clearly presented?

Yes

Are the conclusions supported by the results?

Can be improved

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: Materials and Methods - line 66, "isolation" would be a better term than "extraction". Please, give some details how the cells were isolated from dental pulp tissue.

Response 1: Thank you for this careful comment. We agree with your assessment and have replaced "extraction" with "isolation". We have added a method for the isolation of cells from dental pulp tissue at your suggestion in Page 2, Line 74-81. The revised text reads as follows:

“Teeth were placed in pre-cooled PBS containing 1% P/S immediately after extraction. They were rinsed 3 times with PBS containing 1% P/S to clean the surface. Sterile cotton balls and sterile drapes were used to completely wrap the tooth. A bone mallet was used to split the tooth. As much of the pulp tissue as possible was removed and minced into small pieces. The pulp tissue was digested with 0.1% type I collagenase（BioFroxx）for 1 hour in a 37°C incubator, removed and shaken every 10 minutes. The cell pellet obtained was resuspended and grown in MEM Alpha (Gibco) containing 10% FBS, and 1% penicillin/streptomycin solution. The medium was changed on the fourth day.”

Comments 2: Materials and Methods - line 114, the osteogenic induction only for 3 days is too short to observe significant changes, as the whole osteogenic differentiation takes 21 days.

Response 2: Thank you for your profound questions which help improve the quality of our manuscript. We agree with your assessment that the whole osteogenic differentiation takes 21 days. We only detected alkaline phosphatase activity and gene expression in the early stage of osteogenesis through 3 days of osteogenesis induction. Since we detected the difference within 3 days, so we did not pursuit a longer induction period. In future studies, we will further investigate the effects of replicative senescence and inhibitor-induced senescence on the whole osteogenic differentiation process. Therefore, we have also stated this point in the Discussion section and provided explanations for future research directions in Page 12, Line 337-340. The revised text reads as follows:

“In this study, we only conducted early induction of osteogenesis for 3 days. In the future, we will further examine the effects of replicative senescence and inhibitor-induced senescence on the whole osteogenic differentiation process.”

Comments 3: Materials and Methods - why the Authors did not perform the telomere shortening experiments to confirm the replicative senescence?

Response 3: Thanks for your professional suggestions. We agree with your assessment that telomere shortening is one of the first and best-characterized mechanisms of replicative senescence induction. The focus of our study was on the detection of cellular senescence and not on the detection of replicative senescence. In follow-up studies, we should and will perform telomere-shortening experiments to verify replicative senescence. Therefore, we have also stated this point in the Discussion section and provided explanations for future research directions in Page 12-13, Line 340-342. The revised text reads as follows:

“The detection of replicative senescence in this study mostly focused on cellular senescence. In future studies, we will further use telomere-shortening experiments to verify replicative senescence.”

4. Response to Comments on the Quality of English Language

Point 1: Minor editing of English language required.

Response 1: Thank you for your suggestion. We have tried our best to polish the language and made some changes to the manuscript.

5. Additional clarifications

We would like to express our great appreciation to you for your comments on our paper. And we have tried our best to revise our manuscript according to the comments. Once again, thank you very much for your comments and suggestions. If this article needs further revision, please do not hesitate to contact us.

Response to Reviewer 2 Comments

1. Summary

I would like to express our sincere appreciations of your constructive comments concerning our article entitled “Serine Metabolism Regulates the Replicative Senescence of Human Dental Pulp Cells through Histone Methylation”（ID：cimb-2897366）. These comments are all valuable and helpful for improving our article. According to your comments, we have made extensive modifications to our manuscript. Revised portions are marked in red in the manuscript.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

We have provided our response in the point-by-point response letter. The same as below.

Are all the cited references relevant to the research?

Yes

Is the research design appropriate?

Yes

Are the methods adequately described?

Must be improved

Are the results clearly presented?

Yes

Are the conclusions supported by the results?

Yes

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: 2.1. Cell extraction and culture

It would be helpful if the authors could specify the number of subjects included in the study.

Response 1: Thank you for this careful comment. We have added the number of subjects included in the study according to your suggestion in Page 2, Line 72-73. The revised text reads as follows:

“Four subjects were included: a 22-year-old male, a 23-year-old female, a 24-year-old female, and a 26-year-old female.”

Comments 2: 2.3. Antibody

The authors should provide the catalog numbers of antibodies used in the experiments. It should be specified that actin antibodies, etc., were purchased.

Response 2: Thank you for kindly reminding us. We have added the catalog numbers of antibodies and specified that all antibodies used in the experiments were purchased in Page 3, Line 96-102. The revised text reads as follows:

“All antibodies used in the experiments were purchased. Actin (AF5003, 1:1000) and LMNB1 (AF1408, 1:1000) were purchased from Beyotime; P21 (10355-1-AP, 1:1000) was purchased from Proteintech; PHGDH (66350, 1:1000), yH2AX (9718, 1:800) and Ki67 (9129, 1:400) were purchased from Cell Signaling Technology; PSAT1 (53804, 1:1000) was purchased from Signalway Antibody; PSPH (DF12711, 1:1000) was purchased from Affinity Biosciences; H3 (ab1791, 1:1000), H3K4me3 (ab213224, 1:1000), H3K9me3 (ab176916, 1:1000), H3K27me3 (ab192985, 1:1000 ), H3K36me3 (ab282572, 1:1000) were purchased from Abcam.”

Comments 3: 2.4. RNA extraction and quantitative reverse transcription PCR (RT-qPCR)

The authors should provide the thermal cycle parameters of the qRT-PCR reaction. The term reward sequence in Table 1 should be corrected. The authors should provide the catalog number of the SYBR qPCR Master Mix used.  

Response 3: Thank you for pointing this out. We have added the thermal cycle parameters of the qRT-PCR reaction and the catalog number of the SYBR qPCR Master Mix in Page 3, Line 107-110. We are sorry for our careless mistakes. We have corrected the “reward sequence” into “reverse sequence”. The revised text reads as follows:

“cDNA was quantified using SYBR qPCR Master Mix (Q712-02, Vazyme) under the following conditions: 95 °C for 30 s and then 39 cycles of 95 °C for 10 s and 60 °C for 30 s, followed by a dissociation program at 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 15 s.”

Comments 4: 2.5. S-adenosylmethionine (SAM) assay

The authors should provide the catalog number of the ELISA kit used.

Response 4: Thank you for your suggestion. We have added the catalog number of the ELISA kit used in Page 3, Line 114. The revised text reads as follows:

“The concentration of SAM was detected using an ELISA Kit (CEG414Ge, Cloud-Clone Corp) following the instructions.”

Comments 5: 2.6. Cell Counting Kit-8 (CCK-8) assay

The authors have written: “Cells were treated according to experimental design.” This should be explained. Also, the catalog number of the CCK-8 assay should be provided. A microplate reader and the wavelength of measurement should be specified.

Response 5: Thank you for pointing out this problem. We have re-written this part according to your suggestion in Page 4, Line 117-121. The revised text reads as follows:

“5,000 cells per well were seeded into 96-well plates. Cells were treated with PHGDH inhibitors, NCT-503 and CBR-5884, at different concentrations for 48 hours. After 2-day treatment, 10ul of cck8 (k1018， APExBIO) solution was added per well, then incubated for 1.5 hours. A microplate reader (Multiskan Sky， Thermo Fisher Scientific) was used to read the absorbance at 450 nm.”

Comments 6: 2.8. Colony formation assay

The authors have written: “Cells were treated according to experimental design.” This should be explained.

Response 6: Thank you for reading our manuscript carefully. We have re-written this part according to your suggestion in Page 4, Line 128-130. The revised text reads as follows:

“1,000 cells per well were seeded into 6-well plates. Cells were treated with PHGDH inhibitors (NCT-503 and CBR-5884) for 48 hours or supplemented with serine at different concentrations (0.3mM，1mM) during passage. 0.1% crystal violet was used for staining.”

Comments 7: 2.10. Senescence-associated β-galactosidase (SA-β-gal) staining

The authors have written: “Cells were treated according to experimental design.” This should be explained.

Response 7: Thank you for pointing this out. We have re-written this part according to your suggestion in Page 4, Line 137-140. The revised text reads as follows:

“Cells were seeded into 24-well plates. Cells were treated with PHGDH inhibitors (NCT-503 and CBR-5884) for 48 hours or supplemented with serine at different concentrations (0.3mM，1mM) during passage or treated with both PHGDH inhibitors and serine for 48 hours in serine/glycine-free medium. Senescence β-Galactosidase Staining Kit (Beyotime) was used for staining.”

Comments 8: 2.12. Chromatin immunoprecipitation (ChIP)

The authors should provide the catalog number of the ChIP Assay Kit used.

Response 8: Thank you for your suggestion. We have added the catalog number of the ChIP Assay Kit used in Page 4, Line 150. The revised text reads as follows:

“ChIP was conducted using the ChIP Assay Kit (P2078, Beyotime).”

Comments 9: 2.13. Statistical Analysis

The software used for statistical analysis should be specified.

Response 9: Thank you for your careful comment. We have specified the software at your suggestion in Page 4, Line 158. The revised text reads as follows:

“GraphPad Prism9(v.9.0.0) was used for statistical analysis.”

Comments 10: 3. Results

The meaning of the * in figures should be specified. Obviously, it corresponds to p values smaller than 0.05, but that should be mentioned in figure legends.

Response 10: Thank you for pointing this out. We have specified the meaning of the * in figure legends at your suggestion.

4. Response to Comments on the Quality of English Language

Point 1: A minor revision of the manuscript is suggested.

Response 1: Thank you for your suggestion. We have tried our best to polish the language and made some changes to the manuscript.

5. Additional clarifications

We would like to express our great appreciation to you for your comments on our paper. And we have tried our best to revise our manuscript according to the comments. Once again, thank you very much for your comments and suggestions. If this article needs further revision, please do not hesitate to contact us.

Response to Reviewer 3 Comments

1. Summary

I would like to express our sincere appreciations of your constructive comments concerning our article entitled “Serine Metabolism Regulates the Replicative Senescence of Human Dental Pulp Cells through Histone Methylation” （ID：cimb-2897366）. These comments are all valuable and helpful for improving our article. According to your comments, we have made extensive modifications to our manuscript. Revised portions are marked in red in the manuscript.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Can be improved

We have provided our response in the point-by-point response letter. The same as below.

Are all the cited references relevant to the research?

Yes

Is the research design appropriate?

Must be improved

Are the methods adequately described?

Must be improved

Are the results clearly presented?

Must be improved

Are the conclusions supported by the results?

Can be improved

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: Lack of novelty: The manuscript largely repeats previously reported findings on the individual functions of pathways and molecules involved in cellular senescence. Although the study identifies novel interactions between serine metabolism, histone methylation and expression of specific genes in hDPCs senescence, it fails to provide significant conceptual or methodological innovations. The Introduction and Discussion sections mainly summarise existing knowledge without introducing new hypotheses or insights into the mechanisms underlying hDPCs senescence. Therefore, the authors should take this into account and be more explicit about the existing evidence and the novelty of their own data.

Response 1: Thank you for your feedback and raising the concern regarding the novelty of our study. We appreciate your attention to previously published studies in the field. We would like to address your comment and explain why our study still contributes to the existing knowledge. Although there have been studies on the relationship between cellular senescence of human dental pulp cells (hDPCs) and serine metabolism, we are the first to introduce the concept of tissue engineering. In clinical practice, hDPCs are used in tissue regeneration therapies. However, it requires a large amount of expansion during the application process, which causes the cells to undergo senescence and puts them at risk of developing cancer. Our current results provide evidence that other metabolites derived from the de novo serine synthesis pathway may be involved, rather than serine, in the replicative senescence of hDPCs. In future studies, we will continue to screen for derivatives of serine synthesis pathway that can rescue replicative senescence to delay the senescence of hDPCs during expansion and to improve their efficacy and safety in clinical applications of tissue engineering. The revised text in introduction (Page 2, Line 57-65) and discussion (Page 12, Line 321-332) reads as follows:

Introduction (Page 2, Line 57-65)

“In this study, we investigated for the first time the relationship between serine metabolism and replicative senescence of hDPCs. We demonstrated that hDPCs undergo replicative senescence during passage, and their ability to proliferate and differentiate decreases. During cellular senescence serine metabolism and SAM were decreased, thereby regulating the expression of SIRT1 and RUNX2 through H3K36me3. PHGDH inhibition phenocopied the replicative senescence, both of which involved decreased folate metabolism and could not be rescued by serine supplementation. Our study advances the understanding of replicative senescence and provides a reference for delaying replicative senescence of hDPCs when applied in tissue regeneration therapy.”

Discussion (Page 12, Line 321-332)

“Although there have been studies on the relationship between cellular senescence of hDPCs and serine metabolism, we are the first to introduce the concept of tissue engineering and replicative senescence. As potential seed cells, dental pulp stem cells have been used in tissue engineering and can form the dentine-pulp complex [30], repair bone defects [31,32], and promote periodontal regeneration in infrabony defects [33]. However, it requires a large amount of expansion during the application process, which causes the cells to undergo senescence and puts them at risk of developing cancer [34]. Our current results provide evidence that other metabolites derived from the de novo serine synthesis pathway may be involved, rather than serine, in the replicative senescence of hDPCs. In future studies, we will continue to screen for derivatives of serine synthesis pathway that can rescue replicative senescence to delay the senescence of hDPCs during expansion and to improve their efficacy and safety in clinical applications of tissue engineering.”

References (Page 15, Line 431-444)

16.   Iohara, K.; Imabayashi, K.; Ishizaka, R.; Watanabe, A.; Nabekura, J.; Ito, M.; Matsushita, K.; Nakamura, H.; Nakashima, M. Complete pulp regeneration after pulpectomy by transplantation of CD105+ stem cells with stromal cell-derived factor-1. Tissue engineering. Part A 2011, 17, 1911-1920, doi:10.1089/ten.TEA.2010.0615.

17.   Ito, K.; Yamada, Y.; Nakamura, S.; Ueda, M. Osteogenic potential of effective bone engineering using dental pulp stem cells, bone marrow stem cells, and periosteal cells for osseointegration of dental implants. Int. J. Oral Maxillofac. Implants 2011, 26, 947-954.

18.   Riccio, M.; Maraldi, T.; Pisciotta, A.; La Sala, G.B.; Ferrari, A.; Bruzzesi, G.; Motta, A.; Migliaresi, C.; De Pol, A. Fibroin scaffold repairs critical-size bone defects in vivo supported by human amniotic fluid and dental pulp stem cells. Tissue engineering. Part A 2012, 18, 1006-1013, doi:10.1089/ten.TEA.2011.0542.

19.   Aimetti, M.; Ferrarotti, F.; Cricenti, L.; Mariani, G.M.; Romano, F. Autologous dental pulp stem cells in periodontal regeneration: a case report. Int. J. Periodontics Restorative Dent. 2014, 34 Suppl 3, s27-33, doi:10.11607/prd.1635.

20.   Li, H.; Fan, X.; Kovi, R.C.; Jo, Y.; Moquin, B.; Konz, R.; Stoicov, C.; Kurt-Jones, E.; Grossman, S.R.; Lyle, S.; et al. Spontaneous expression of embryonic factors and p53 point mutations in aged mesenchymal stem cells: a model of age-related tumorigenesis in mice. Cancer Res. 2007, 67, 10889-10898, doi: 10.1158/0008-5472.can-07-2665.

Comments 2: Inadequate description of qPCR analyses: The manuscript makes extensive use of quantitative polymerase chain reaction (qPCR) analyses to assess gene expression levels in hDPCs senescence. However, the methodology and data analysis procedures for qPCR are poorly described. It is important to specify whether the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines were followed to ensure the quality and accuracy of the qPCR data.

Response 2: We would like to thank you for your professional review work and constructive comments. We have re-written this part according to your suggestion. And we declare that all procedures follow the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. The revised text in Page 3, Line 105-111 reads as follows:

“RNA was isolated using TRIzol (Invitrogen) and its absorbance was measured with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). Reverse Transcriptase Kit (Vazyme) was used to synthesize cDNA. cDNA was quantified using SYBR qPCR Master Mix system (Q712-02, Vazyme) under the following conditions: 95 °C for 30 s and then 39 cycles of 95 °C for 10 s and 60 °C for 30 s, followed by a dissociation program at 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 15 s. β-actin was used as an internal reference. The 2-ΔΔCt method was used to quantify the levels of gene expression. Primers are listed in Table 1.”

Comments 3: Questionable statistical analyses: The manuscript uses parametric statistical analyses to assess the significance of experimental results. However, given the small sample size (n=3) in the experimental groups, the use of parametric tests is highly questionable. Parametric tests assume normal distribution and homogeneity of variance, which may not be valid with such a small sample size. Therefore, the authors need to reconsider the choice of statistical tests and provide a rationale for their selection based on the assumptions and limitations of the experimental design.

Response 3: Thank you for your profound questions which help improve the quality of our manuscript. We think this is an excellent suggestion. We regret that we were not clear enough in describing the statistical methods used. Before selecting statistical methods, we first performed normal distribution and homogeneity of variance tests on the data. According to statistical knowledge, when the requirements of parametric statistics are met, that is, when conditions such as normality and homogeneity of variances are met, the test efficiency of parametric statistics is higher than that of nonparametric statistics, and parametric statistics is preferred. In our study, the Shapiro-Wilk test was first used to test normality. For two groups of data, if normality and homogeneity of variances are satisfied, an unpaired t-test is used. If normality is not satisfied or variances are not equal, the Mann-Whitney U test is used. The data in three or more groups in our study are all in accordance with the normal distribution and the homogeneity of the variances. For these datas, one-way/two-way ANOVA is used, and multiple comparisons are performed using the Turkey test. We apologize again for the unclear description and have added the appropriate description in the text. The revised text in Page 4-5, Line 159-164 reads as follows:

“The Shapiro-Wilk test was used first to test for normality. For two groups of data, if normality and homogeneity of variances are satisfied, an unpaired t-test is used. If normality is not satisfied or variances are not equal, the Mann-Whitney U test is used. For three or more groups of data that all satisfy normality and homogeneity of variances, one-way/two-way ANOVA is used, and multiple comparisons are performed using the Turkey test.”

4. Response to Comments on the Quality of English Language

Point 1: Moderate editing of English language required.

Response 1: Thank you for your suggestion. We have tried our best to polish the language and made some changes to the manuscript.

5. Additional clarifications

We would like to express our great appreciation to you for your comments on our paper. And we have tried our best to revise our manuscript according to the comments. Once again, thank you very much for your comments and suggestions. If this article needs further revision, please do not hesitate to contact us.