The combination of ADSCs and 10% PRP increases Rb protein expression on senescent human dermal fibroblasts

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Since adipose-derived stem cells (ADSCs) have a paracrine effect, ADSCs were utilized to improve the senescence process of HDFs. The use of non-autologous cell culture media to grow ADSCs can be legally problematic; therefore, platelet-rich plasma (PRP) can be considered as an alternative medium. PRP contains various growth factors that can be used to process the reversal of senescent HDFs. The combination of ADSCs and PRP is expected to increase the expression of Rb protein in HDFs that have undergone the senescence process. Methods: This study was performed in vitro with a randomized sample, and non-blinded pre-and post-test control group. The primary culture of senescent HDFs was transfected with a combination of ADSCs and 10% PRP. The effect on migration was observed through the scratch test, while the effect of PRP on reversal senescence was observed through Sa-β-gal analysis and the expression of protein Rb with ELISA. Results: The senescent HDFs that received a combined transfection of ADSCs and 10% PRP proliferated rapidly in the scratch test. Based on the Sa-β-gal assay, they showed fewer senescent HDFs cells. The combination of ADSCs and 10% PRP elevated the expression of Rb protein significantly (P < 0.001). Conclusions: The combination of ADSCs and 10% PRP was shown to have a reversal effect on the senescence process of HDFs in vitro." } { "@context": "http://schema.org", "@type": "BreadcrumbList", "itemListElement": [ { "@type": "ListItem", "position": "1", "item": { "@id": "https://f1000research.com/", "name": "Home" } }, { "@type": "ListItem", "position": "2", "item": { "@id": "https://f1000research.com/browse/articles", "name": "Browse" } }, { "@type": "ListItem", "position": "3", "item": { "@id": "https://f1000research.com/articles/10-516/v1", "name": "The combination of ADSCs and 10% PRP increases Rb protein expression..." } } ] } Home Browse The combination of ADSCs and 10% PRP increases Rb protein expression... ALL Metrics - Views Downloads Get PDF Get XML Cite How to cite this article Murlistyarini S, Aninda LP, Afridafaz UA et al. The combination of ADSCs and 10% PRP increases Rb protein expression on senescent human dermal fibroblasts [version 1; peer review: 1 approved, 1 not approved] . F1000Research 2021, 10 :516 ( https://doi.org/10.12688/f1000research.53170.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Research Article The combination of ADSCs and 10% PRP increases Rb protein expression on senescent human dermal fibroblasts [version 1; peer review: 1 approved, 1 not approved] Sinta Murlistyarini https://orcid.org/0000-0001-5706-8610 1,2 , Lulus Putri Aninda https://orcid.org/0000-0002-6845-4477 3 , Ufida Aini Afridafaz 3 , Sri Widyarti 1,4 , Agustina Tri Endharti https://orcid.org/0000-0002-2062-5740 1,5,6 , Teguh Wahju Sardjono 1,5 Sinta Murlistyarini https://orcid.org/0000-0001-5706-8610 1,2 , Lulus Putri Aninda https://orcid.org/0000-0002-6845-4477 3 , [...] Ufida Aini Afridafaz 3 , Sri Widyarti 1,4 , Agustina Tri Endharti https://orcid.org/0000-0002-2062-5740 1,5,6 , Teguh Wahju Sardjono 1,5 PUBLISHED 30 Jun 2021 Author details Author details 1 Doctoral Program of Medical Science, Faculty of Medicine, Universitas Brawijaya, Malang, 65145, Indonesia 2 Department of Dermatology and Venerology, Faculty of Medicine, Universitas Brawijaya, Malang, 65145, Indonesia 3 Faculty of Medicine, Universitas Brawijaya, Malang, 65145, Indonesia 4 Department of Biology, Universitas Brawijaya, Malang, 65145, Indonesia 5 Department of Parasitology, Universitas Brawijaya, Malang, 65145, Indonesia 6 Biomedical Sciences, Faculty of Medicine, Universitas Brawijaya, Malang, 65145, Indonesia Sinta Murlistyarini Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing Lulus Putri Aninda Roles: Formal Analysis, Investigation, Methodology, Writing – Review & Editing Ufida Aini Afridafaz Roles: Formal Analysis, Investigation, Methodology Sri Widyarti Roles: Formal Analysis, Investigation, Supervision, Writing – Review & Editing Agustina Tri Endharti Roles: Formal Analysis, Supervision, Writing – Review & Editing Teguh Wahju Sardjono Roles: Formal Analysis, Funding Acquisition, Investigation, Supervision, Writing – Review & Editing OPEN PEER REVIEW DETAILS REVIEWER STATUS This article is included in the Cell & Molecular Biology gateway. This article is included in the Advances in Fibroblast Research collection. Abstract Background: The senescence process in human dermal fibroblasts (HDFs) is caused by cell cycle withdrawal processes, one of which is the result of the retinoblastoma (Rb) protein being in a hypo-phosphorylated state. Since adipose-derived stem cells (ADSCs) have a paracrine effect, ADSCs were utilized to improve the senescence process of HDFs. The use of non-autologous cell culture media to grow ADSCs can be legally problematic; therefore, platelet-rich plasma (PRP) can be considered as an alternative medium. PRP contains various growth factors that can be used to process the reversal of senescent HDFs. The combination of ADSCs and PRP is expected to increase the expression of Rb protein in HDFs that have undergone the senescence process. Methods: This study was performed in vitro with a randomized sample, and non-blinded pre-and post-test control group. The primary culture of senescent HDFs was transfected with a combination of ADSCs and 10% PRP. The effect on migration was observed through the scratch test, while the effect of PRP on reversal senescence was observed through Sa-β-gal analysis and the expression of protein Rb with ELISA. Results: The senescent HDFs that received a combined transfection of ADSCs and 10% PRP proliferated rapidly in the scratch test. Based on the Sa-β-gal assay, they showed fewer senescent HDFs cells. The combination of ADSCs and 10% PRP elevated the expression of Rb protein significantly ( P < 0.001). Conclusions: The combination of ADSCs and 10% PRP was shown to have a reversal effect on the senescence process of HDFs in vitro . READ ALL READ LESS Keywords Cellular senescence, human dermal fibroblasts, adipose-derived stem cells, platelet-rich plasma, protein retinoblastoma Corresponding Author(s) Sinta Murlistyarini ( [email protected] ) Close Corresponding author: Sinta Murlistyarini Competing interests: No competing interests were disclosed. Grant information: This study was supported by the Institute of Research and Community Services Universitas Brawijaya (Contract Number 1/UN10.F08/PN/2019). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2021 Murlistyarini S et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Murlistyarini S, Aninda LP, Afridafaz UA et al. The combination of ADSCs and 10% PRP increases Rb protein expression on senescent human dermal fibroblasts [version 1; peer review: 1 approved, 1 not approved] . F1000Research 2021, 10 :516 ( https://doi.org/10.12688/f1000research.53170.1 ) First published: 30 Jun 2021, 10 :516 ( https://doi.org/10.12688/f1000research.53170.1 ) Latest published: 30 Jun 2021, 10 :516 ( https://doi.org/10.12688/f1000research.53170.1 ) Introduction Adipose-derived stem cells (ADSCs) are mesenchymal stem cells that reside in fat tissue and were first identified by Zuk et al. 1 ADSCs with a unique secretome have a paracrine effect on surrounding cells giving rise to the theory that the efficacy of stem cell therapy is more due to the paracrine effect of cell secretion than the effect of cell differentiation. 2 The paracrine effect of ADSCs is widely used in regenerative medicine such as tissue repair, wound healing, and as an anti-aging therapy. 3 Conditioned media from adipose-derived stem cells (ADSC-CM) contains all the bioactive components of ADSCs, and is easy to store and safer during application. 4 The cells are often cultured using a basal medium containing foetal bovine serum (FBS). FBS is important in the cell culture process. However, when it is used for regenerative medicine, this serum is less safe because it contains xenoproteins, which can cause rejection or transmission of infectious products. As a result, nowadays, the use of non-autologous cell culture media, like FBS in ADSCs for clinical applications is legally challenged. Thus, to reduce the use of animal products in ADSC cultures, a study was conducted using platelet-rich plasma (PRP) instead of FBS. 25 The use of PRP is intended as a safer alternative supplement medium for ADSC culture. PRP is blood plasma that contains many platelets which are often used for wound healing. 5 Platelets are a natural source of various growth factors such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), endothelial growth factor (EGF), and epidermal growth factor (EGF). They are efficient at triggering cell proliferation, differentiation, and tissue regeneration. 6 , 7 There are many protocols for PRP preparation, from conventional blood centrifugation to other innovative methods, and the PRP can be activated by adding other materials or methods, for example, calcium, collagen, and/thrombin, or by glass contact or freeze-thaw cycles. 8 , 9 Cellular senescence is an arrest of the cell cycle with a phenotypic change in which the cell will lose its replicative ability. 10 In the past, cellular senescence was considered an irreversible process as protection against cancer, however, recent findings have shown its role in aging, tissue repair and development of senescent fibroblasts. 11 In vitro , cells that experience senescence will undergo morphological changes such as cell flattening and enlargement, the apparition of vacuoles, and sometimes multiple nuclei; these contribute significantly to the aging process. 12 - 14 Cells that experience senescence will lower the expression of genes involved in the cell cycle and several components of the extracellular matrix, and increase the expression of cell cycle inhibitor genes and matrix-degrading enzymes. 15 Cell cycle withdrawal associated with cellular senescence is triggered by the p53-p21 and p16-Rb pathways. 16 , 17 In senescent human dermal fibroblasts (HDFs), retinoblastoma (Rb) proteins accumulate in an active state; a hypo-phosphorylation state. In this condition, this protein is unable to phosphorylate in the middle or the end of the G1 phase and thus it is unable to enter the S phase. 18 Research by Song et al . showed that ADSCs decreased p16 expression in senescent fibroblasts, thus it appears that ADSCs can reverse the aging process. 19 Several extensive studies on PRP have been conducted, however, the biological mechanisms and clinical effects of PRP on HDFs are unclear. Besides, there has been no research on the combination of ADSC-CM and 10% PRP on Rb protein in senescent HDFs. Methods Our research has been done at the Biomedical and Parasitology laboratory, Faculty of Medicine, Universitas Brawijaya, Malang Indonesia. The HDFs as a culture source for fibroblast cells and ADSCs were isolated from a 25-year-old pregnant woman during an elective caesarean operation. The participant provided informed written consent prior to the study and all of the procedures in this research were approved by the Ethics Committee of the Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia (No.169/EC/KEPK-S3/05/2019). The HDF cells were an explant culture from human skin and were put into a 6-well plate until the explant attached. The cells were grown in a solution containing 20% FBS and Dulbecco’s Modified Eagle Medium (FBS-DMEM, ThermoFisher). The method used was a modified version of the method used by Takashima (1998), while the senescent HDF method used was that of Radiono et al. (2016). 20 , 21 The HDFs were shifted into a senescent state by replacing the medium with 10% PRP in DMEM (ThermoFisher). ADSCs from the pregnant woman were cultivated in 6-well plates and cultured in a complete culture medium (10% PRP in DMEM). The PRP preparation mentioned above was adopted from Amable et al. 22 Briefly, whole blood was placed in tubes containing anticoagulants, and was centrifuged for 20 min (3,000 × g ). After obtaining three layers in the whole blood, the upper layer was transferred into 10 ml empty sterile micro centrifuge tubes. The PRP was activated with 20mM CaCl 2 . The expression of senescence-associated β-galactosidase (Sa- β -Gal) was analysed in HDFs using purchased Sa- β -Gal staining kit (MyBioSource, San Diego, CA, CAT# MBS168501 ) referring to manufacturer’s procedures. Concisely, the cells were fixed with 25% glutaraldehyde for 5 min, then they were washed with phosphate-buffered saline (PBS). Subsequently, the cells were incubated in cell staining solution overnight. The stained HDFs were washed with PBS and examined using an IX71 inverted microscope (Olympus) at 200× magnification. The migration of HDF cells was investigated using a scratch assay. HDFs were grown to 80% confluence in 24-well plates and scraped with a 200 μl pipette tip. Next, the media were replaced to reduce the debris. HDF cells were photographed at 0 and 72h thereafter. After that, the HDFs were washed with PBS and detached by trypsin. The cell suspension was collected and centrifuged for 5 min (1000×g). The measurement of Rb protein levels was done using an enzyme-linked immunosorbent assay kit (ELISA) according to the protocol issued by the manufacturer’s instruction (MyBioSource, Cat# MBS2509425 , RRID: AB_10568804). In brief, 100 μl of standard or sample were added to each well and incubated for 90 min at 37°C. The liquid was replaced with the provided biotinylated monoclonal antibody (60 min, 37°C) and followed by washing 3 times. The biotinylated detection antibody is specific to Human Rb1 and Avidin-Horseradish Peroxidase (HRP) conjugate. A 100 μl HRP conjugate was added (30 min, 37°C). After washing 5 times, 90 μl of substrate reagent was added and incubated for 15 min at 37°C. The reaction was stopped with 50 μl stop solution and the optical density (OD) was directly determined at 450 nm. The data obtained was analysed using SPSS software version 25 (IBM). The normality test was carried out by the Shapiro Wilk test. If the P-value >0.05, then the normality assumption was fulfilled so an independent t-test was carried out. However, if the normality test was not fulfilled, the Mann-Whitney test was performed. Results The 10% PRP - DMEM media was used to culture ADSCs and these media were used to shift the senescence state in HDFs. HDF explant culture began to grow on the seventh day. After the seventh passage, the HDF culture medium was replaced with 0.5% FBS for 72 hours to allow HDF cells to undergo senescence. The HDF media was then removed and the cells were transfected with the ADSCs - 10% PRP for another 72 h and subsequently examined by ELISA to determine the Rb protein level. The scratch test showed that the senescent HDF cell group that received the ADSCs – 10% PRP medium proliferated faster than the control group ( Figure 1 ). The Sa-β-Gal investigation showed that cells with ADSCs – 10% PRP medium had a lower number of senescent cells than the control group ( Figure 2 ). Figure 1. Scratch test results. Scratch test of HDFs in the control group on day 0 (A) and after 72 hours (B). Scratch test of HDFs in the ADSCs – 10% PRP group on day 0 (C) and after 72 hours (D). ADSCs: adipose-derived stem cells; HDF: human dermal fibroblasts; PRP: platelet-rich plasma. Figure 2. Sa-β-Gal staining of fibroblasts in the control group (A) and senescent fibroblasts treated with ADSCs – 10% PRP (B). ADSCs, adipose-derived stem cells; PRP, platelet-rich plasma. Based on the Shapiro Wilk normality test, the P-value obtained for both groups was > 0.05, thus the independent t-test was carried out and gave a statistically significant result ( P < 0.001) ( Table 1 , 38 ). Table 1. The results of the independent t-test of Rb protein expression. Group Average Rb protein expression ± SD (ng/ml) P value Control 0.04 ± 0.012 <0.001 ADSCs – 10% PRP 0.127 ± 0.049 Discussion Besides being easy to develop in vitro , ADSCs have similar capabilities to bone marrow stem cells, but they can be obtained easily from subcutaneous adipose tissue without invasive action, can maintain concentrations and proliferation consistently, and represent a more biologically relevant model for studying aging mechanisms when compared to other stem cells. 23 , 24 PRP is a fraction of autologous blood plasma with a high platelet concentration. 26 PRP also produces various cytokines, chemokines, and growth factors that can trigger recruitment, adhesion, proliferation, and maintain the differentiation of ADSCs. 25 , 27 Research by Jia et al. (2017), which examined the effect of PRP in an in vitro photo-aging model, showed that PRP reduced the number of aging cells by 1% based on Sa-β-Gal positive cell tests. Furthermore, PRP prevents cell cycle arrest caused by irradiation by reducing the expression of p53 and p21, and decreasing the expression of matrix metalloproteinase (MMP) -1, -3, and -9 but not MMP-2 at the mRNA level. 28 Research conducted by Kocaoemer et al. (2007) stated that 10% of non-autologous PRP activated by shock freezing or thrombin after being cultured for 11 days will increase ADSC proliferation faster than 10% FBS. 29 Meanwhile, Pham et al. (2013) found that 15% PRP is the optimal concentration that will stimulate ADSC proliferation. 30 Also, using the supernatant of the 10% activated PRP increases the proliferation of mesenchymal stem cells compared to other supplementary media such as whole blood, non-activated PRP, and non-activated platelet-poor plasma. 31 Higher PRP concentrations will interfere with cell growth in vitro because the platelets will release protein complexes that negatively affect the action of the PRP growth factor. 32 Besides, the concentration of proteolytic enzymes such as collagenase, cathepsin, elastase, and acid phosphatase is very high in PRP; therefore, it causes inhibition of cell growth. 33 Stessuk et al. (2016) studied the combination of PRP with ADSC-CM in vitro and found significant proliferation stimulation in fibroblasts cultured with 25% PRP combined with ADSC-CM after 48 hours, while keratinocyte proliferation also increased insignificantly after 24 hours. 34 Park et al. (2011) found that 10% PRP stimulates fibroblast cell migration and proliferation within 24 hours, presumably because PRP releases the PDGF-AA growth factor. However, PRP did not have a significant effect on the activity of MMP-1, MMP-2, and MMP-9 on HDFs cells. This is appropriate because fibroblast cells will be strongly reactive to PDGF, b-FGF, and EGF. 35 Liu et al. (2014) examined the ability of PRP to improve cellular conditions from the senescence process and subsequently be able to slow down aging in animals. The research showed that PRP can change the expression of senescence markers such as p16, p19, and p53 on transgenic mice. In addition, PRP also can trigger cell growth, proliferation, and colony formation, and increase osteogenesis, reduce adipogenesis, and fight oxidative stress in stem cells from aged mice. 36 Cho et al. (2012) examined the effects of PRP on cell migration, proliferation, and expression of various cell cycle regulatory proteins in HDFs cells. From this study, it was found that low concentrations of PRP (0.05 – 0.5%) would increase the expression of cyclin-A in HDFs cells, and 5% PRP would enhance the expression of proteins involved in the cell cycle, for example, Rb protein, cyclin-E, cyclin-A, and Cdk2. It seems that PRP through various growth factors will promote the progression of the cell cycle back into the G1 phase by regulating the expression level and activation of cyclin-Cdks. 37 In summary, this study found an increase in Rb protein expression on senescent HDFs which were given a combination of ADSCs and 10% PRP. However, further studies are still needed to determine the effect of the combination of ADSCs and PRP on other senescence markers. Data availability Underlying data Figshare: Supplementary Data - The combination of ADSCs and 10% PRP increase Rb protein expression on senescent human dermal fibroblasts, https://doi.org/10.6084/m9.figshare.14716314.v1 . 38 This project contains the following underlying data: - The combination of ADSCs and 10% PRP increase Rb protein expression on senescent human dermal fibroblasts.xlsx - Statistic Result The combination of ADSCs and 10% PRP increase Rb protein expression on senescent human dermal fibroblasts.docx - Picture 1A. Scratch test of HDFs in the control group on day 0.jpeg - Picture 1B. Scratch test of HDFs in the control group after 72 h.jpeg - Picture 1C. Scratch test of HDFs in the ADSCs – 10% PRP group on day 0.jpeg - Picture 1D. Scratch test of HDFs in the ADSCs – 10% PRP group after 72 h.jpeg - Picture 2A. SaBGal staining of fibroblasts in the control group.jpeg - Picture 2B. SaBGal staining of senescent fibroblasts treated with ADSCs – 10% PRP.jpeg - Figure 1.jpeg - Figure 2.jpeg Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0) . Acknowledgments The authors would like to thank RSIA Melati Husada Malang, Indonesia, for providing the fat and dermal tissue. The authors would like to thank Dr. Tinny Endang Hernowati, SpPK (K), for her kindness and support during the research, may she Rest In Peace. References 1. Zuk P: Adipose-derived stem cells in tissue regeneration: a review, ISRN Stem Cells.2013 Article ID 713959. Publisher Full Text 2. Lai RC, Yeo RWY, Lim SK: Mesenchymal stem cell exosomes. Semin Cell Dev Biol. 2015; 40 : 82–88. PubMed Abstract | Publisher Full Text 3. Shen X, Du Y, Shen W, et al. : Adipose-derived stem cells promote human dermal fibroblast function and increase senescence-associated-β-galactosidase mRNA expression through paracrine effects. Mol Med Rep. 2014; 10 : 3068–3072. 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Dataset. 2021. Publisher Full Text Comments on this article Comments (1) Version 1 VERSION 1 PUBLISHED 30 Jun 2021 Reader Comment 23 Jul 2021 Anita Bandrowski , SciCrunch Inc, USA, USA 23 Jul 2021 Reader Comment I ran a SciScore.com report for this preprint to highlight which types of rigor criteria were addressed by the authors. Please note, the items specified below are listed on the ... Continue reading I ran a SciScore.com report for this preprint to highlight which types of rigor criteria were addressed by the authors. Please note, the items specified below are listed on the MDAR reproducibility checklist (and others). For a full description of the criteria tested for please take a look at the FAQs at the bottom of the report. SciScore: 6/10 Document Identifier: 102249 SciScore Report Below you will find your SciScore report containing three tables. Your score is calculated based on adherence to scientific rigor criteria (Table 1) and identification of key biological resources (Table 2). Table 3 contains statistical tests and oligonucleotides but is not scored. If SciScore makes any mistakes, please contact us to help us learn and improve. Table 1: Rigor Adherence Table Ethics Consent: The participant provided informed written consent prior to the study and all of the procedures in this research were approved by the Ethics Committee of the Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia (No.169/EC/KEPK-S3/05/2019). IRB: The participant provided informed written consent prior to the study and all of the procedures in this research were approved by the Ethics Committee of the Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia (No.169/EC/KEPK-S3/05/2019). Inclusion and Exclusion Criteria not detected. Attrition not detected. Sex as a biological variable The HDFs as a culture source for fibroblast cells and ADSCs were isolated from a 25-year-old pregnant woman during an elective caesarean operation. Subject Demographics Age: The HDFs as a culture source for fibroblast cells and ADSCs were isolated from a 25-year-old pregnant woman during an elective caesarean operation. Randomization not detected. Blinding not detected. Power Analysis not detected. Replication not required. Table 2: Key Resources Table Antibodies The measurement of Rb protein levels was done using an enzyme-linked immunosorbent assay kit (ELISA) according to the protocol issued by the manufacturer’s instruction (MyBioSource, Cat#MBS2509425, RRID: AB_10568804). RRID Verified: (MyBioSource Cat# MBS120046, RRID:AB_10568804)(link ) Software and Algorithms The data obtained was analysed using SPSS software version 25 (IBM). SPSS RRID Suggestion: (SPSS, RRID:SCR_002865)( link) Table 3 Other Entities Detected Your Sentences Recognized Entity Statistical Tests The normality test was carried out by the Shapiro Wilk test. Entity detected: Wilk test If the P-value >0.05, then the normality assumption was fulfilled so an independent t-test was carried out. Entity detected: t-test However, if the normality test was not fulfilled, the Mann-Whitney test was performed. Entity detected: Mann-Whitney test Please note: SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore is not a substitute for expert review. SciScore also checks for the presence and correctness of several unique identifiers, including RRIDs (research resource identifiers) in the manuscript, detects sentences that appear to be missing RRIDs, and can even suggest RRIDs under certain circumstances. All RRID suggestions should be verified; only the author can know whether the suggestions are correct. For a full description of scored criteria and tips for improving your score, please see https://www.scicrunch.com/sciscorereport-faq I ran a SciScore.com report for this preprint to highlight which types of rigor criteria were addressed by the authors. Please note, the items specified below are listed on the MDAR reproducibility checklist (and others). For a full description of the criteria tested for please take a look at the FAQs at the bottom of the report. SciScore: 6/10 Document Identifier: 102249 SciScore Report Below you will find your SciScore report containing three tables. Your score is calculated based on adherence to scientific rigor criteria (Table 1) and identification of key biological resources (Table 2). Table 3 contains statistical tests and oligonucleotides but is not scored. If SciScore makes any mistakes, please contact us to help us learn and improve. Table 1: Rigor Adherence Table Ethics Consent: The participant provided informed written consent prior to the study and all of the procedures in this research were approved by the Ethics Committee of the Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia (No.169/EC/KEPK-S3/05/2019). IRB: The participant provided informed written consent prior to the study and all of the procedures in this research were approved by the Ethics Committee of the Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia (No.169/EC/KEPK-S3/05/2019). Inclusion and Exclusion Criteria not detected. Attrition not detected. Sex as a biological variable The HDFs as a culture source for fibroblast cells and ADSCs were isolated from a 25-year-old pregnant woman during an elective caesarean operation. Subject Demographics Age: The HDFs as a culture source for fibroblast cells and ADSCs were isolated from a 25-year-old pregnant woman during an elective caesarean operation. Randomization not detected. Blinding not detected. Power Analysis not detected. Replication not required. Table 2: Key Resources Table Antibodies The measurement of Rb protein levels was done using an enzyme-linked immunosorbent assay kit (ELISA) according to the protocol issued by the manufacturer’s instruction (MyBioSource, Cat#MBS2509425, RRID: AB_10568804). RRID Verified: (MyBioSource Cat# MBS120046, RRID:AB_10568804)(link ) Software and Algorithms The data obtained was analysed using SPSS software version 25 (IBM). SPSS RRID Suggestion: (SPSS, RRID:SCR_002865)( link) Table 3 Other Entities Detected Your Sentences Recognized Entity Statistical Tests The normality test was carried out by the Shapiro Wilk test. Entity detected: Wilk test If the P-value >0.05, then the normality assumption was fulfilled so an independent t-test was carried out. Entity detected: t-test However, if the normality test was not fulfilled, the Mann-Whitney test was performed. Entity detected: Mann-Whitney test Please note: SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore is not a substitute for expert review. SciScore also checks for the presence and correctness of several unique identifiers, including RRIDs (research resource identifiers) in the manuscript, detects sentences that appear to be missing RRIDs, and can even suggest RRIDs under certain circumstances. All RRID suggestions should be verified; only the author can know whether the suggestions are correct. For a full description of scored criteria and tips for improving your score, please see https://www.scicrunch.com/sciscorereport-faq Competing Interests: I am a creator of SciScore Close Report a concern Comment ADD YOUR COMMENT Author details Author details 1 Doctoral Program of Medical Science, Faculty of Medicine, Universitas Brawijaya, Malang, 65145, Indonesia 2 Department of Dermatology and Venerology, Faculty of Medicine, Universitas Brawijaya, Malang, 65145, Indonesia 3 Faculty of Medicine, Universitas Brawijaya, Malang, 65145, Indonesia 4 Department of Biology, Universitas Brawijaya, Malang, 65145, Indonesia 5 Department of Parasitology, Universitas Brawijaya, Malang, 65145, Indonesia 6 Biomedical Sciences, Faculty of Medicine, Universitas Brawijaya, Malang, 65145, Indonesia Sinta Murlistyarini Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing Lulus Putri Aninda Roles: Formal Analysis, Investigation, Methodology, Writing – Review & Editing Ufida Aini Afridafaz Roles: Formal Analysis, Investigation, Methodology Sri Widyarti Roles: Formal Analysis, Investigation, Supervision, Writing – Review & Editing Agustina Tri Endharti Roles: Formal Analysis, Supervision, Writing – Review & Editing Teguh Wahju Sardjono Roles: Formal Analysis, Funding Acquisition, Investigation, Supervision, Writing – Review & Editing Competing interests No competing interests were disclosed. Grant information This study was supported by the Institute of Research and Community Services Universitas Brawijaya (Contract Number 1/UN10.F08/PN/2019). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Article Versions (1) version 1 Published: 30 Jun 2021, 10:516 https://doi.org/10.12688/f1000research.53170.1 Copyright © 2021 Murlistyarini S et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Murlistyarini S, Aninda LP, Afridafaz UA et al. The combination of ADSCs and 10% PRP increases Rb protein expression on senescent human dermal fibroblasts [version 1; peer review: 1 approved, 1 not approved] . F1000Research 2021, 10 :516 ( https://doi.org/10.12688/f1000research.53170.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 1 VERSION 1 PUBLISHED 30 Jun 2021 Views 0 Cite How to cite this report: Harkness T and Lubachowski M. Reviewer Report For: The combination of ADSCs and 10% PRP increases Rb protein expression on senescent human dermal fibroblasts [version 1; peer review: 1 approved, 1 not approved] . F1000Research 2021, 10 :516 ( https://doi.org/10.5256/f1000research.56525.r279795 ) The direct URL for this report is: https://f1000research.com/articles/10-516/v1#referee-response-279795 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 06 Jun 2024 Troy Harkness , University of Alberta, Edmonton, AB, Canada Mathew Lubachowski , Medicine, University of Alberta, Edmonton, Alberta, Canada Not Approved VIEWS 0 https://doi.org/10.5256/f1000research.56525.r279795 This article addresses the issue of cellular senescence, and the role it plays in processes such as aging. The authors test whether treatment with adipose-derived stem cells (ADSC) in combination with 10% platelet rich plasma (PRP) can reverse the senescent ... Continue reading READ ALL This article addresses the issue of cellular senescence, and the role it plays in processes such as aging. The authors test whether treatment with adipose-derived stem cells (ADSC) in combination with 10% platelet rich plasma (PRP) can reverse the senescent phenotype in human dermal fibroblasts (HDF). While the basic study design shows promise, there are several issues that need addressing to ensure validity of the research, and the correct interpretation of results. Likewise, improvement of the article organization and grammar, as well as the inclusion of additional details within the text, will ensure the readers ability to understand the article, methods, and analysis. Please address the following points. A transcript of the text with suggested edits for improving its spelling/grammar and readability is available upon request. Abstract: “The primary culture of senescent HDFs was transfected with a combination of ADSCs and 10% PRP.” Transfection refers to transferring DNA into a mammalian cell. I believe the authors meant that the HDFs and ADSCs were combined in a single culture with the addition of PRP. This was observed again in the Results section of the abstract. Also, some grammar issues in the abstract were observed. In the first paragraph of the Results section, it is again stated that “The HDF media was then removed and the cells were transfected with the ADSCs - 10% PRP for another 72 h”. Transfected is not correct. If the term transfected is being used for another purpose, then please explain. It is explained that this was done in order to use ELISA to determine Rb levels. The authors then move on to the scratch test and never state what levels the Rb protein were at. Why use ELISA and not simply a Western? It seems a western would be easier, quicker and less expensive. As the introduction is quite short, I believe the authors could easily include a sentence or two of background regarding the relationship between p16 and RB. Simply stating that p16 is an inhibitor of CDKs responsible for Rb phosphorylation will frame the role of p16, allowing better understanding for readers unfamiliar with the pathway when the authors state that “Research by Song et al . showed that ADSCs decreased p16 expression in senescent fibroblasts, thus it appears that ADSCs can reverse the aging process” in paragraph 4 of the introduction. I believe that paragraphs 3 and 4 of the introduction should be moved to the beginning, ahead of paragraphs 1 and 2. Paragraphs 3 and 4 introduce the issue that the authors are trying to address, and as such should be discussed first to provide context for the reader. The ELISA conducted measured total Rb protein, an increase in which is interpreted by the authors as evidence of a reduction in senescence. Would measurement of phospho-Rb, and potentially a comparison of the amount of phospho-Rb to total Rb, be a more appropriate indicator? In relation to point 5 the authors presented only quantification of Rb protein, but there was no normalization to account for variance in total protein expression, or cell density. I would suggest that the authors use western blots instead of ELISA. Using western blotting the authors will be able to more easily compare the expression of total and phosphorylated Rb, as well as normalize the Rb data to housekeeping genes and/or total protein amount. Furthermore, by resolving the lysates by size it will help confirm specificity of the antibody signal. It was unclear to me what the control cell treatment was. Please clarify this in the methods and/or figure legends. Was the control maintenance of serum starvation, or return to 20% FBS? I believe it is important for the authors to include a control of 20% FBS to show that the effect seen in the treatment is not simply a reversal of serum starvation when cells are given an alternate to serum (PRP). Similarly, the authors should include treatments where cells are treated with PRP alone, or ADSC conditioned media with FBS instead of PRP. This will allow the authors to show whether the combination of PRP and ADSC conditioned media has an additive effect, and how much of the effect is resultant from which component, or if the total effect is reliant on only one of the two. Building on point 7, I believe it would provide much better support for the authors finding if they were to replicate them using a model of senescence other than serum starvation. Induction of senescence using UV or H 2 O 2 are potential techniques that have been used in human HDFs (Gerasymchuk et al., 2022; Yao et al., 2022). The methods for lysate preparation were not clear. Please clarify. It is hard for the reader to interpret figure one. Could the contrast be improved? Results should also be quantified by either measuring the portion of the wound that has closed, or by quantifying the migration distance of each cell in time lapsed images (Cory, 2011). The scratch test measures how fast a void in a cell lawn can be filled by cells. Typically, a time course over 24-48 hours is conducted with images taken every 12 hours or so. It isn’t a measure of proliferation, but a measure of migration. By 72 hours, migration is typically complete, so this experiment, which images cells at 0 and 72 hours, simply measures how many cells entered the void and says nothing regarding cell proliferation. The images shown appear to have more cells in the void in the control cells, so their conclusions are not supported by the images. The images should be quantified showing the numbers of cells that enter the void from at least 3 independent experiments that show a time course. It is not stated how many times this was repeated, so it is likely it was only done once. The data should be shown in graph form complete with statistical analyses, by either measuring the portion of the wound that has closed, or by quantifying the migration distance of each cell in time lapsed images (Cory, 2011). Quantification should also be done on Sa-β-Gal staining. Counting the ratio of stained to total cells would be a simple method of this. When discussing Sa-β-Gal staining in the results the author should make it clear that this is being used as a senescence marker. The b-gal assay appears to show that there is more staining in the control group, but the image is low resolution and there is a lot of background, with nothing labelled, so it is unclear what is being shown in this image. This really isn’t a clean experiment. As mentioned above, a western should also be done to validate the ELISA. We do not know the quality of the antibody used, so have no idea what is really being bound. Paragraph 3 of the results should be moved to follow paragraph 1, as this would improve the flow of the article. The authors could also comment more on the observed differences between treatment and control groups rather than just the significance of said differences. What was the number of replicates for the ELISA? State this in methods and figure legend. The discussion reads as a listing of previous findings by other research groups. The authors should use the discussion to address and draw conclusions from their own results and use previous findings from the literature to frame, support, or contrast their own conclusions. In conclusion, this is an incredibly simple paper with very little experimentation that lacks complete controls and validation experiments. The conclusions drawn are not easily seen from the poor quality of the images shown. This paper is not suitable for indexing in its present form. 1) Cory, G. (2011 [Ref -1]). Scratch-Wound Assay. Methods in Molecular Biology , 769 , 25–30. https://doi.org/10.1007/978-1-61779-207-6_2 2) Gerasymchuk, M., et al. (2022 [Ref - 2]). Modeling of the Senescence-Associated Phenotype in Human Skin Fibroblasts. International Journal of Molecular Sciences , 23 (13). https://doi.org/10.3390/IJMS23137124 3) Yao, X., et al. (2022 [Ref -3]). UV-induced senescence of human dermal fibroblasts restrained by low-stiffness matrix by inhibiting NF-í µí¼ B activation. Engineered Regeneration , 3 , 365–373. https://doi.org/10.1016/j.engreg.2022.08.002 Is the work clearly and accurately presented and does it cite the current literature? No Is the study design appropriate and is the work technically sound? No Are sufficient details of methods and analysis provided to allow replication by others? No If applicable, is the statistical analysis and its interpretation appropriate? No Are all the source data underlying the results available to ensure full reproducibility? No source data required Are the conclusions drawn adequately supported by the results? No References 1. Cory G: Scratch-wound assay. Methods Mol Biol . 2011; 769 : 25-30 PubMed Abstract | Publisher Full Text 2. Gerasymchuk M, Robinson GI, Kovalchuk O, Kovalchuk I: Modeling of the Senescence-Associated Phenotype in Human Skin Fibroblasts. Int J Mol Sci . 2022; 23 (13). PubMed Abstract | Publisher Full Text 3. Yao X, Li H, Chen L, Tan L: UV-induced senescence of human dermal fibroblasts restrained by low-stiffness matrix by inhibiting NF-κB activation. Engineered Regeneration . 2022; 3 (4): 365-373 Publisher Full Text Competing Interests: No competing interests were disclosed. Reviewer Expertise: Yeast and human aging and cancer, cell cycle regulation, and ubiquitin signalling. We confirm that we have read this submission and believe that we have an appropriate level of expertise to state that we do not consider it to be of an acceptable scientific standard, for reasons outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Harkness T and Lubachowski M. Reviewer Report For: The combination of ADSCs and 10% PRP increases Rb protein expression on senescent human dermal fibroblasts [version 1; peer review: 1 approved, 1 not approved] . F1000Research 2021, 10 :516 ( https://doi.org/10.5256/f1000research.56525.r279795 ) The direct URL for this report is: https://f1000research.com/articles/10-516/v1#referee-response-279795 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Views 0 Cite How to cite this report: MAWARDI P and Fitri LE. Reviewer Report For: The combination of ADSCs and 10% PRP increases Rb protein expression on senescent human dermal fibroblasts [version 1; peer review: 1 approved, 1 not approved] . F1000Research 2021, 10 :516 ( https://doi.org/10.5256/f1000research.56525.r90976 ) The direct URL for this report is: https://f1000research.com/articles/10-516/v1#referee-response-90976 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 20 Aug 2021 PRASETYADI MAWARDI , Department of Dermatology and Venereology, Faculty of Medicine, Sebelas Maret University, Surakarta, Indonesia; Dr. Moewardi General Hospital, Surakarta, Indonesia Loeki Enggar Fitri , Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia Approved VIEWS 0 https://doi.org/10.5256/f1000research.56525.r90976 This study presents the results of how the senescence process is not an irreversible process, which will spur other studies in the mystery of the cellular senescence process. The role of PRP and ADSC in increasing the migration process of ... Continue reading READ ALL This study presents the results of how the senescence process is not an irreversible process, which will spur other studies in the mystery of the cellular senescence process. The role of PRP and ADSC in increasing the migration process of human dermal fibroblast cells has proven that cellular senescence is a dynamic, continuous process that will be useful in aging therapy in the future. This research is also carefully structured, coherent, methodological, and has adequate analysis. In some respects, Sa-β-Gal, has its own characteristics as a parameter of cellular senescence. Determination of the Rb protein is an important biomarker in the cell cycle and is the right choice to support the research excellence. I suggest adding the following statement so that there is a relationship between the biomarker senescence-associated β-galactosidase (Sa-β-Gal) activity and Rb protein in the introduction: " Cellular senescence is a cell cycle arrest process that accompanies depletion of replicative potential in normal somatic cell cultures. The senescent cells are characterized by several molecular and cytological markers, such as an enlarged and flattened morphology and the expression of senescence-associated β-galactosidase (Sa-β-Gal) activity. A large body of evidence suggests that tumor suppressor pathways Rb and p53 are key regulators of cellular senescence." My suggestion can be included in the introduction before the last paragraph, which can certainly add to the strength of this research, because I noticed in the introductory section the problem of Sa-β-Gal and Rb protein was very little explained; whereas Sa-β-Gal and Rb proteins are the main objects of research. To support this statement I also include the reference that I use (Campisi J: Senescent cells, tumor suppression, and organismal aging: good citizens, bad neighbors. Cell. 2005; 120 (4): 513-22 1 ). For keywords : 'Cellular senescence, human dermal fibroblasts, adipose-derived stem cells, platelet-rich plasma, protein retinoblastoma' .. protein retinoblastoma can be replaced by retinoblastoma protein . Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes References 1. Campisi J: Senescent cells, tumor suppression, and organismal aging: good citizens, bad neighbors. Cell . 2005; 120 (4): 513-22 PubMed Abstract | Publisher Full Text Competing Interests: No competing interests were disclosed. Reviewer Expertise: Clinical dermatology, venereology, skin cancer, biomolecular, dermatopathology We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT MAWARDI P and Fitri LE. Reviewer Report For: The combination of ADSCs and 10% PRP increases Rb protein expression on senescent human dermal fibroblasts [version 1; peer review: 1 approved, 1 not approved] . F1000Research 2021, 10 :516 ( https://doi.org/10.5256/f1000research.56525.r90976 ) The direct URL for this report is: https://f1000research.com/articles/10-516/v1#referee-response-90976 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Comments on this article Comments (1) Version 1 VERSION 1 PUBLISHED 30 Jun 2021 Reader Comment 23 Jul 2021 Anita Bandrowski , SciCrunch Inc, USA, USA 23 Jul 2021 Reader Comment I ran a SciScore.com report for this preprint to highlight which types of rigor criteria were addressed by the authors. Please note, the items specified below are listed on the ... Continue reading I ran a SciScore.com report for this preprint to highlight which types of rigor criteria were addressed by the authors. Please note, the items specified below are listed on the MDAR reproducibility checklist (and others). For a full description of the criteria tested for please take a look at the FAQs at the bottom of the report. SciScore: 6/10 Document Identifier: 102249 SciScore Report Below you will find your SciScore report containing three tables. Your score is calculated based on adherence to scientific rigor criteria (Table 1) and identification of key biological resources (Table 2). Table 3 contains statistical tests and oligonucleotides but is not scored. If SciScore makes any mistakes, please contact us to help us learn and improve. Table 1: Rigor Adherence Table Ethics Consent: The participant provided informed written consent prior to the study and all of the procedures in this research were approved by the Ethics Committee of the Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia (No.169/EC/KEPK-S3/05/2019). IRB: The participant provided informed written consent prior to the study and all of the procedures in this research were approved by the Ethics Committee of the Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia (No.169/EC/KEPK-S3/05/2019). Inclusion and Exclusion Criteria not detected. Attrition not detected. Sex as a biological variable The HDFs as a culture source for fibroblast cells and ADSCs were isolated from a 25-year-old pregnant woman during an elective caesarean operation. Subject Demographics Age: The HDFs as a culture source for fibroblast cells and ADSCs were isolated from a 25-year-old pregnant woman during an elective caesarean operation. Randomization not detected. Blinding not detected. Power Analysis not detected. Replication not required. Table 2: Key Resources Table Antibodies The measurement of Rb protein levels was done using an enzyme-linked immunosorbent assay kit (ELISA) according to the protocol issued by the manufacturer’s instruction (MyBioSource, Cat#MBS2509425, RRID: AB_10568804). RRID Verified: (MyBioSource Cat# MBS120046, RRID:AB_10568804)(link ) Software and Algorithms The data obtained was analysed using SPSS software version 25 (IBM). SPSS RRID Suggestion: (SPSS, RRID:SCR_002865)( link) Table 3 Other Entities Detected Your Sentences Recognized Entity Statistical Tests The normality test was carried out by the Shapiro Wilk test. Entity detected: Wilk test If the P-value >0.05, then the normality assumption was fulfilled so an independent t-test was carried out. Entity detected: t-test However, if the normality test was not fulfilled, the Mann-Whitney test was performed. Entity detected: Mann-Whitney test Please note: SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore is not a substitute for expert review. SciScore also checks for the presence and correctness of several unique identifiers, including RRIDs (research resource identifiers) in the manuscript, detects sentences that appear to be missing RRIDs, and can even suggest RRIDs under certain circumstances. All RRID suggestions should be verified; only the author can know whether the suggestions are correct. For a full description of scored criteria and tips for improving your score, please see https://www.scicrunch.com/sciscorereport-faq I ran a SciScore.com report for this preprint to highlight which types of rigor criteria were addressed by the authors. Please note, the items specified below are listed on the MDAR reproducibility checklist (and others). For a full description of the criteria tested for please take a look at the FAQs at the bottom of the report. SciScore: 6/10 Document Identifier: 102249 SciScore Report Below you will find your SciScore report containing three tables. Your score is calculated based on adherence to scientific rigor criteria (Table 1) and identification of key biological resources (Table 2). Table 3 contains statistical tests and oligonucleotides but is not scored. If SciScore makes any mistakes, please contact us to help us learn and improve. Table 1: Rigor Adherence Table Ethics Consent: The participant provided informed written consent prior to the study and all of the procedures in this research were approved by the Ethics Committee of the Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia (No.169/EC/KEPK-S3/05/2019). IRB: The participant provided informed written consent prior to the study and all of the procedures in this research were approved by the Ethics Committee of the Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia (No.169/EC/KEPK-S3/05/2019). Inclusion and Exclusion Criteria not detected. Attrition not detected. Sex as a biological variable The HDFs as a culture source for fibroblast cells and ADSCs were isolated from a 25-year-old pregnant woman during an elective caesarean operation. Subject Demographics Age: The HDFs as a culture source for fibroblast cells and ADSCs were isolated from a 25-year-old pregnant woman during an elective caesarean operation. Randomization not detected. Blinding not detected. Power Analysis not detected. Replication not required. Table 2: Key Resources Table Antibodies The measurement of Rb protein levels was done using an enzyme-linked immunosorbent assay kit (ELISA) according to the protocol issued by the manufacturer’s instruction (MyBioSource, Cat#MBS2509425, RRID: AB_10568804). RRID Verified: (MyBioSource Cat# MBS120046, RRID:AB_10568804)(link ) Software and Algorithms The data obtained was analysed using SPSS software version 25 (IBM). SPSS RRID Suggestion: (SPSS, RRID:SCR_002865)( link) Table 3 Other Entities Detected Your Sentences Recognized Entity Statistical Tests The normality test was carried out by the Shapiro Wilk test. Entity detected: Wilk test If the P-value >0.05, then the normality assumption was fulfilled so an independent t-test was carried out. Entity detected: t-test However, if the normality test was not fulfilled, the Mann-Whitney test was performed. Entity detected: Mann-Whitney test Please note: SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore is not a substitute for expert review. SciScore also checks for the presence and correctness of several unique identifiers, including RRIDs (research resource identifiers) in the manuscript, detects sentences that appear to be missing RRIDs, and can even suggest RRIDs under certain circumstances. All RRID suggestions should be verified; only the author can know whether the suggestions are correct. For a full description of scored criteria and tips for improving your score, please see https://www.scicrunch.com/sciscorereport-faq Competing Interests: I am a creator of SciScore Close Report a concern Comment ADD YOUR COMMENT keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 Version 1 30 Jun 21 read read PRASETYADI MAWARDI , Sebelas Maret University, Surakarta, Indonesia; Dr. Moewardi General Hospital, Surakarta, Indonesia Loeki Enggar Fitri , Universitas Brawijaya, Malang, Indonesia Troy Harkness , University of Alberta, Edmonton, AB, Canada Mathew Lubachowski , University of Alberta, Edmonton, Canada Comments on this article All Comments (1) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2024 Harkness T et al. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 06 Jun 2024 | for Version 1 Troy Harkness , University of Alberta, Edmonton, AB, Canada Mathew Lubachowski , Medicine, University of Alberta, Edmonton, Alberta, Canada 0 Views copyright © 2024 Harkness T et al. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Not Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions This article addresses the issue of cellular senescence, and the role it plays in processes such as aging. The authors test whether treatment with adipose-derived stem cells (ADSC) in combination with 10% platelet rich plasma (PRP) can reverse the senescent phenotype in human dermal fibroblasts (HDF). While the basic study design shows promise, there are several issues that need addressing to ensure validity of the research, and the correct interpretation of results. Likewise, improvement of the article organization and grammar, as well as the inclusion of additional details within the text, will ensure the readers ability to understand the article, methods, and analysis. Please address the following points. A transcript of the text with suggested edits for improving its spelling/grammar and readability is available upon request. Abstract: “The primary culture of senescent HDFs was transfected with a combination of ADSCs and 10% PRP.” Transfection refers to transferring DNA into a mammalian cell. I believe the authors meant that the HDFs and ADSCs were combined in a single culture with the addition of PRP. This was observed again in the Results section of the abstract. Also, some grammar issues in the abstract were observed. In the first paragraph of the Results section, it is again stated that “The HDF media was then removed and the cells were transfected with the ADSCs - 10% PRP for another 72 h”. Transfected is not correct. If the term transfected is being used for another purpose, then please explain. It is explained that this was done in order to use ELISA to determine Rb levels. The authors then move on to the scratch test and never state what levels the Rb protein were at. Why use ELISA and not simply a Western? It seems a western would be easier, quicker and less expensive. As the introduction is quite short, I believe the authors could easily include a sentence or two of background regarding the relationship between p16 and RB. Simply stating that p16 is an inhibitor of CDKs responsible for Rb phosphorylation will frame the role of p16, allowing better understanding for readers unfamiliar with the pathway when the authors state that “Research by Song et al . showed that ADSCs decreased p16 expression in senescent fibroblasts, thus it appears that ADSCs can reverse the aging process” in paragraph 4 of the introduction. I believe that paragraphs 3 and 4 of the introduction should be moved to the beginning, ahead of paragraphs 1 and 2. Paragraphs 3 and 4 introduce the issue that the authors are trying to address, and as such should be discussed first to provide context for the reader. The ELISA conducted measured total Rb protein, an increase in which is interpreted by the authors as evidence of a reduction in senescence. Would measurement of phospho-Rb, and potentially a comparison of the amount of phospho-Rb to total Rb, be a more appropriate indicator? In relation to point 5 the authors presented only quantification of Rb protein, but there was no normalization to account for variance in total protein expression, or cell density. I would suggest that the authors use western blots instead of ELISA. Using western blotting the authors will be able to more easily compare the expression of total and phosphorylated Rb, as well as normalize the Rb data to housekeeping genes and/or total protein amount. Furthermore, by resolving the lysates by size it will help confirm specificity of the antibody signal. It was unclear to me what the control cell treatment was. Please clarify this in the methods and/or figure legends. Was the control maintenance of serum starvation, or return to 20% FBS? I believe it is important for the authors to include a control of 20% FBS to show that the effect seen in the treatment is not simply a reversal of serum starvation when cells are given an alternate to serum (PRP). Similarly, the authors should include treatments where cells are treated with PRP alone, or ADSC conditioned media with FBS instead of PRP. This will allow the authors to show whether the combination of PRP and ADSC conditioned media has an additive effect, and how much of the effect is resultant from which component, or if the total effect is reliant on only one of the two. Building on point 7, I believe it would provide much better support for the authors finding if they were to replicate them using a model of senescence other than serum starvation. Induction of senescence using UV or H 2 O 2 are potential techniques that have been used in human HDFs (Gerasymchuk et al., 2022; Yao et al., 2022). The methods for lysate preparation were not clear. Please clarify. It is hard for the reader to interpret figure one. Could the contrast be improved? Results should also be quantified by either measuring the portion of the wound that has closed, or by quantifying the migration distance of each cell in time lapsed images (Cory, 2011). The scratch test measures how fast a void in a cell lawn can be filled by cells. Typically, a time course over 24-48 hours is conducted with images taken every 12 hours or so. It isn’t a measure of proliferation, but a measure of migration. By 72 hours, migration is typically complete, so this experiment, which images cells at 0 and 72 hours, simply measures how many cells entered the void and says nothing regarding cell proliferation. The images shown appear to have more cells in the void in the control cells, so their conclusions are not supported by the images. The images should be quantified showing the numbers of cells that enter the void from at least 3 independent experiments that show a time course. It is not stated how many times this was repeated, so it is likely it was only done once. The data should be shown in graph form complete with statistical analyses, by either measuring the portion of the wound that has closed, or by quantifying the migration distance of each cell in time lapsed images (Cory, 2011). Quantification should also be done on Sa-β-Gal staining. Counting the ratio of stained to total cells would be a simple method of this. When discussing Sa-β-Gal staining in the results the author should make it clear that this is being used as a senescence marker. The b-gal assay appears to show that there is more staining in the control group, but the image is low resolution and there is a lot of background, with nothing labelled, so it is unclear what is being shown in this image. This really isn’t a clean experiment. As mentioned above, a western should also be done to validate the ELISA. We do not know the quality of the antibody used, so have no idea what is really being bound. Paragraph 3 of the results should be moved to follow paragraph 1, as this would improve the flow of the article. The authors could also comment more on the observed differences between treatment and control groups rather than just the significance of said differences. What was the number of replicates for the ELISA? State this in methods and figure legend. The discussion reads as a listing of previous findings by other research groups. The authors should use the discussion to address and draw conclusions from their own results and use previous findings from the literature to frame, support, or contrast their own conclusions. In conclusion, this is an incredibly simple paper with very little experimentation that lacks complete controls and validation experiments. The conclusions drawn are not easily seen from the poor quality of the images shown. This paper is not suitable for indexing in its present form. 1) Cory, G. (2011 [Ref -1]). Scratch-Wound Assay. Methods in Molecular Biology , 769 , 25–30. https://doi.org/10.1007/978-1-61779-207-6_2 2) Gerasymchuk, M., et al. (2022 [Ref - 2]). Modeling of the Senescence-Associated Phenotype in Human Skin Fibroblasts. International Journal of Molecular Sciences , 23 (13). https://doi.org/10.3390/IJMS23137124 3) Yao, X., et al. (2022 [Ref -3]). UV-induced senescence of human dermal fibroblasts restrained by low-stiffness matrix by inhibiting NF-í µí¼ B activation. Engineered Regeneration , 3 , 365–373. https://doi.org/10.1016/j.engreg.2022.08.002 Is the work clearly and accurately presented and does it cite the current literature? No Is the study design appropriate and is the work technically sound? No Are sufficient details of methods and analysis provided to allow replication by others? No If applicable, is the statistical analysis and its interpretation appropriate? No Are all the source data underlying the results available to ensure full reproducibility? No source data required Are the conclusions drawn adequately supported by the results? No References 1. Cory G: Scratch-wound assay. Methods Mol Biol . 2011; 769 : 25-30 PubMed Abstract | Publisher Full Text 2. Gerasymchuk M, Robinson GI, Kovalchuk O, Kovalchuk I: Modeling of the Senescence-Associated Phenotype in Human Skin Fibroblasts. Int J Mol Sci . 2022; 23 (13). PubMed Abstract | Publisher Full Text 3. Yao X, Li H, Chen L, Tan L: UV-induced senescence of human dermal fibroblasts restrained by low-stiffness matrix by inhibiting NF-κB activation. Engineered Regeneration . 2022; 3 (4): 365-373 Publisher Full Text Competing Interests No competing interests were disclosed. Reviewer Expertise Yeast and human aging and cancer, cell cycle regulation, and ubiquitin signalling. We confirm that we have read this submission and believe that we have an appropriate level of expertise to state that we do not consider it to be of an acceptable scientific standard, for reasons outlined above. reply Respond to this report Responses (0) Harkness T and Lubachowski M. Peer Review Report For: The combination of ADSCs and 10% PRP increases Rb protein expression on senescent human dermal fibroblasts [version 1; peer review: 1 approved, 1 not approved] . F1000Research 2021, 10 :516 ( https://doi.org/10.5256/f1000research.56525.r279795) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/10-516/v1#referee-response-279795 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2021 MAWARDI P et al. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 20 Aug 2021 | for Version 1 PRASETYADI MAWARDI , Department of Dermatology and Venereology, Faculty of Medicine, Sebelas Maret University, Surakarta, Indonesia; Dr. Moewardi General Hospital, Surakarta, Indonesia Loeki Enggar Fitri , Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia 0 Views copyright © 2021 MAWARDI P et al. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions This study presents the results of how the senescence process is not an irreversible process, which will spur other studies in the mystery of the cellular senescence process. The role of PRP and ADSC in increasing the migration process of human dermal fibroblast cells has proven that cellular senescence is a dynamic, continuous process that will be useful in aging therapy in the future. This research is also carefully structured, coherent, methodological, and has adequate analysis. In some respects, Sa-β-Gal, has its own characteristics as a parameter of cellular senescence. Determination of the Rb protein is an important biomarker in the cell cycle and is the right choice to support the research excellence. I suggest adding the following statement so that there is a relationship between the biomarker senescence-associated β-galactosidase (Sa-β-Gal) activity and Rb protein in the introduction: " Cellular senescence is a cell cycle arrest process that accompanies depletion of replicative potential in normal somatic cell cultures. The senescent cells are characterized by several molecular and cytological markers, such as an enlarged and flattened morphology and the expression of senescence-associated β-galactosidase (Sa-β-Gal) activity. A large body of evidence suggests that tumor suppressor pathways Rb and p53 are key regulators of cellular senescence." My suggestion can be included in the introduction before the last paragraph, which can certainly add to the strength of this research, because I noticed in the introductory section the problem of Sa-β-Gal and Rb protein was very little explained; whereas Sa-β-Gal and Rb proteins are the main objects of research. To support this statement I also include the reference that I use (Campisi J: Senescent cells, tumor suppression, and organismal aging: good citizens, bad neighbors. Cell. 2005; 120 (4): 513-22 1 ). For keywords : 'Cellular senescence, human dermal fibroblasts, adipose-derived stem cells, platelet-rich plasma, protein retinoblastoma' .. protein retinoblastoma can be replaced by retinoblastoma protein . Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes References 1. Campisi J: Senescent cells, tumor suppression, and organismal aging: good citizens, bad neighbors. Cell . 2005; 120 (4): 513-22 PubMed Abstract | Publisher Full Text Competing Interests No competing interests were disclosed. Reviewer Expertise Clinical dermatology, venereology, skin cancer, biomolecular, dermatopathology We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) MAWARDI P and Fitri LE. Peer Review Report For: The combination of ADSCs and 10% PRP increases Rb protein expression on senescent human dermal fibroblasts [version 1; peer review: 1 approved, 1 not approved] . F1000Research 2021, 10 :516 ( https://doi.org/10.5256/f1000research.56525.r90976) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. 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