Reversine enhances the conversion of dedifferentiated fat cells into mature cardiomyocytes

Background There is an essential need for cardiomyocyte regeneration among patients with heart failure. Transplantation of dedifferentiated fat (DFAT) cells may lead to an improvement of cardiomyocyte regeneration among heart failure patients. We believe that DFAT cells are promising candidate cell sources for cardiac regeneration. However, the pathway underlying how DFAT cells of the adipose lineage differentiate into mature cardiomyocytes isn’t fully understood. Methods We conducted an experimental laboratory study on isolated DFAT cells from adipose tissue of healthy adults. Then, we treated cells with different concentrations of reversine (10, 20 and 40 nM), and performed RNA extraction and cDNA synthesis. Next, we used a ceiling culture method based on the buoyancy properties of mature lipid-filled adipocytes. Stemness expression (Octamer-binding transcription factor 4 [Oct4], brachyury, Fetal liver kinase 1 [Flk-1]) was quantified by reverse transcription-quantitative (RT-q)PCR, while cardiomyocyte expression (Transcription factor GATA-4 [GATA4] and cardiac troponin T [cTnT]) was quantified by immunocytochemistry. Results ANOVA with Tukey’s post-hoc found that 10 nM reversine increased greater Flk-1 expression compared to the control group (MD: 5.037 + 0.998; p < 0.001), but there were no significant changes among Oct4 (MD: 0.013 + 1.244; p = 0.99) and brachyury expression (MD: 0.157 + 0.084; p = 0.252). Kruskal-Wallis revealed that the expression of GATA4 (1.65 [0.41-1.98] to 0.015 [0.007-0.034]; p =0.017) reduced significantly from day 7 until day 21 and cTnT (5.07 [6.62-8.91] to 8.22 [6.81-9.40]; p= 0 .001) increased significantly from day 7 until day 21. Conclusions Reversine could increase the expression of Flk-1, but it was unable to stimulate the expression of Oct4 and brachyury related to cell stemness. An optimal concentration of 10 nM reversine may have the greatest effect on enhancing the differentiation of DFAT cells into mature cardiomyocytes, as indicated by higher cTnT expression between cells.


Background
There is an essential need for cardiomyocyte regeneration among patients with heart failure.Transplantation of dedifferentiated fat (DFAT) cells may lead to an improvement of cardiomyocyte regeneration among heart failure patients.We believe that DFAT cells are promising candidate cell sources for cardiac regeneration.However, the pathway underlying how DFAT cells of the adipose lineage differentiate into mature cardiomyocytes isn't fully understood.

Methods
We conducted an experimental laboratory study on isolated DFAT cells from adipose tissue of healthy adults.Then, we treated cells with different concentrations of reversine (10, 20  Any reports and responses or comments on the article can be found at the end of the article. ceiling culture method based on the buoyancy properties of mature lipid-filled adipocytes.Stemness expression (Octamer-binding transcription factor 4 [Oct4], brachyury, Fetal liver kinase 1 [Flk-1]) was quantified by reverse transcription-quantitative (RT-q)PCR, while cardiomyocyte expression (Transcription factor GATA-4 [GATA4] and cardiac troponin T [cTnT]) was quantified by immunocytochemistry.

Conclusions
Reversine could increase the expression of Flk-1, but it was unable to stimulate the expression of Oct4 and brachyury related to cell stemness.An optimal concentration of 10 nM reversine may have the greatest effect on enhancing the differentiation of DFAT cells into mature cardiomyocytes, as indicated by higher cTnT expression between cells.

Introduction
Heart failure is a complex health problem due to its high morbidity and mortality, so a definite and efficient approach to heart failure is a long-term hope and goal in the development of heart disease therapy.The population of those with heart failure increases as the number of older individuals grows and life expectancy generally increases due to the advances in pharmacology, interventions, and cardiac surgery.Classic interventions focus on controlling factors that might worsen cardiac function.These interventions do not solve the real problem, because widespread and progressive cardiomyocyte dysfunction has still taken place.Thus, novel approaches that modify the remodelling process, focus on the regeneration of cardiomyocytes, and improve the performance of cardiomyocytes are required. 1generative medicine combines an interdisciplinary research and clinical application that aims to repair, replace, and/or regenerate cells, tissues and/or organs to restore disturbed function. 1,2These approaches include, but are not limited, to the use of small and/or soluble molecule, gene therapy, stem cell-based therapy, organ tissue engineering, and

REVISED Amendments from Version 2
-There is slight rephrase of the title -There are several grammatical improvement -Merged and shortened sections of Aim / Objectives -There is correction for several symbols such as μ which is missing -Correction for several discrepancies between text and table -Merged and deleted several subsections in the Results -Information regarding the use of 6 th cell passage treatments for data analysis and figures -Correction for Figure 2. caption -Correction for Figure 2, 3, 4, and 5 regarding the standard deviation bars and p values clearer, as a comparison between each group of intervention, not between day -Additional citation and references number 7 [Mitrečić D et al., 2022]   Any further responses from the reviewers can be found at the end of the article reprogramming of cells. 3,4As a precursor for stem cell harvesting in cell-based therapy, adipose tissue has unique advantages in terms of abundant availability, ease of isolation, high degree of homogeneity, and decent ability to differentiate into multipotent cells. 5,6The identification of an ideal cell source for tissue regeneration remains a challenge in the stem cell field. 7With the current development of isolation and culture techniques, ceiling culture and insert culture will produce adipose-derived stem cells (ASCs) with a higher quantity of dedifferentiated fat (DFAT) cells. 8,9milar to ASCs, DFAT cells have been proven to be able to differentiate into various cells in the mesenchymal lineage. 10,11DFAT cells have plasticity to differentiate into several cell types derived from the three germ layers, including skeletal myoblasts, 12 vascular endothelial cells, 13 neurons, 14 and urethral smooth muscle cells. 15AT cells are a population of cells which can be safely harvested from patients.Due to their low immunogenicity and reported benefits, they are already being recognized as approved therapeutic product.They were demonstrated to be safe but their effects were not always consistent, as preclinical studies suggested. 7This may be due to poor survival in disease environments and/or their limited ability to differentiate into other cell types, which is important for the processes of tissue reconstruction and tissue engineering and has clinical, biochemical or molecular implications. 7versine is a purine-derived small molecule that has been shown to be able to induce the dedifferentiation of unipotent myoblast cells to progenitor cells, which are multipotent. 16,17Reversine as an inductor of cell dedifferentiation that has the ability to increase the potency of a cell, so that the cell can be directed to differentiate into another cell in the same lineage or even from a different lineage with exposure to the appropriate stimulus and medium. 18,19 date there has been no research that proves the effect of reversine on the process of further dedifferentiation of adipocyte cells, which causes a reduction in the commitment lineage so that it can be relatively more easily directed towards target cells, so this study aims to analyse the effect of reversine exposure and differences in passages of DFAT cells related to their differentiation into cardiomyocytes, characterized by the expression of the marker Transcription factor GATA-4 (GATA4) and cardiac troponin T (cTnT).We performed a pilot study to understand the optimal concentration of reversine to maximise the potency of DFAT cell dedifferentiation into cardiac progenitor stem cells.We used different concentrations of reversine (10, 20 and 40 nM) in the DFAT cell cultures.

Ethical approval
Our study obtained a letter of approval from Health Research Ethics Council of Faculty of Medicine, Universitas Airlangga (reference number: 062/EC/KEPK/FKUA/2021) issued on March 22 nd 2021, under the name of Budi Baktijasa Dharmadjati as the principal investigator.All procedures were approved by the relevant ethics committees, and written informed consent was obtained from all study participants.

Study design
The study design is an observational, analytical laboratory study using different concentrations of reversine in DFAT cell cultures from human adipose tissue from a 39 years-old woman.This type of analytical laboratory study is a post-test only controlled group design.The independent variables consist of various concentrations of reversine (10, 20, 40 nM).The dependent variables consist of the expression of cell stemness biomarkers (Octamer-binding transcription factor 4 [Oct4], brachyury, Fetal liver kinase 1 [Flk-1]), early cardiomyocytes (GATA4) and mature cardiomyocytes (cTnT).

Study setting
Our research was conducted at Institute of Stem Cell Laboratory Airlangga University, Suraaya, East Java and Dr. Soetomo General Academic Hospital, Surabaya, East Java.The duration between study enrolment until data completion lasted for 12 months.

Number of replications
The number of replicates that will produce this interval half-length according to Berthouex and Brown (2002)  20 is: This formula assumes random sampling.It also assumes that n is large enough that the normal distribution can be used to define the confidence interval.Then, the number of replications for each group is four, so the total sample needed is 16 replications.

Materials
1. DFAT cells from adipose tissue were collected from the lower abdominal area of patients during laparoscopic surgery with small incisions (3-5 cm) under local anaesthesia by a digestive surgeon.Cells were collected from adult patients who were in a stable condition and were not taking anti-platelets or anti-coagulants.Patient was prepared for clinical application of stem cell therapy at the Network Bank Dr. Soetomo General Hospital, Surabaya.DFAT cells were multiplied in vitro at the 3 rd and 6 th passage.

Collection and isolation of DFAT cells from adipose tissue
Adipose tissue obtained from loose subcutaneous tissue through abdominoplasty procedure with a size of ~3x3 cm with an estimated weight of 10 g and stored in a 50 mL conical tube.Adipose tissue was minced into small pieces and dissociated with 0.1% (w/v) collagenase, then sent to the laboratory using a container containing an ice pack at a temperature of 4°C without the addition of other cryopreservation agents.When the tissue was received in the laboratory, the adipocyte tissue was put into a 50 mL plastic tube to be washed with D-PBS (-) twice at room temperature.The tissue was then placed into a 10 cm glass petri dish, then chopped until smooth.
Insert culture strategy was performed to collect mature lipid-filled adipocytes based on the property of buoyancy.Approximately 1 g adipose tissue was chopped and mixed into 0.1% collagenase solution (type I collagenase) at 37°C for 1 hour with stirring/shaking slowly.After centrifugation at 135 Â g for 3 minutes, the supernatant layer containing DFAT cells floated on the culture medium or plastic tube to allow other non-adipocytes to separate and sink to the bottom and can be discarded after centrifugation, then filtered using a nylon filter (core size 100 m).Adipose was then washed repeatedly (3 times) in MEM supplemented with 20% foetal bovine serum (FBS) before further use.A total of 30 µL was then transferred to 6-well plates with 70 µm filters and incubated for 5 days in MEM.DFAT cells from adipocytes will sink through the filter and stick to the bottom of the dish.The filter and residual adipocytes were discarded after day 5.
Cells were treated with different concentrations of reversine (10, 20 and 40 nM).Reversine (StemCell Technologies) at a dose of 10, 20 and 40 nM was administered to DFAT cell sub-groups passage 3 and 6 on the first day of DFAT cells entering the passage.Then, DFAT cells were incubated for 15 minutes at room temperature.

RNA extraction and cDNA synthesis
For extraction of RNA, single cell pellets from each well in the first group were transferred to a conical tube for further centrifugation at 13,000 rpm for 3 minutes (Wisespin CF10, WISD).The pellets formed were separated and 200 μL liquid was left in the tube.Pellets were rinsed with PBS, then 560 µL prepared Buffer AVL containing carrier RNA (QiAmp ® Viral RNA Kit) were added into a 1.5 mL microcentrifuge tube.Vortexing (Wisemix VM-10, WISD) was performed for 15 seconds followed by spin down (Vision VS-100 BN) before incubation at room temperature for 15 minutes.After the incubation process was completed, 600 μL of 96% ethanol was added to the sample tube.Vortexing was again performed for 15 seconds followed by spin down.The solution in the tube was then transferred to a spin column and centrifuged at 8,000 rpm for 1 minute.After the supernatant layer was removed, the contents of the tube were transferred to a 2 mL collection tube.Wash buffer AW1 was added to the collection tube before centrifugation at 8,000 rpm for 1 minute.After rinsing with wash buffer 2, the supernatant layer was removed and centrifuged at 12,000 rpm for 1 minute in order to dry the filter, with a target concentration in the filter of 1-2 ng/μL.Then the solution was transferred to a 1.5 mL tube and 60 μL AVE buffer was added.After incubation at room temperature for 3 minutes, centrifugation was carried out at 8,000 rpm for 1 minute.The solution was then stored in a collecting tube in a cooler at -20°C.
For cDNA synthesis, the RNA samples and reagents were thawed at room temperature and the PCR tubes were prepared and labelled.Vortexing was carried out to ensure that the solution mixture was homogeneous, and a spin down process was carried out to ensure that all liquids remained at the bottom.A total of 2 µL reagent was added to the PCR tube, then 3 µL RNA sample was added to the same PCR tube.After vortexing and spin down, the next tube was inserted into the PCR machine at 70°C for 5 minutes.cDNAs were generated from 1 mg total RNA and amplified using the ReverTra Ace qPCR-RT Kit (Toyobo, Osaka, Japan) according to the manufacturer's instructions.

Reverse transcription-quantitative (RT-q)PCR
Quantification of cell stemness expression (Oct4, brachyury, Flk-1) was performed via RT-qPCR.Primer sequences of Oct4, brachyury and Flk-1 can be seen in Table 1.Primer reagent (0.5 μL) with nuclease-free water (1.5 μL) were vortexed and span down at 70°C for 5 minutes.Another reagent consisted of 4 μL Go Script buffer, 4 μL MgCl 2 , 1 μL PCR Nucleotides, 1 μL Reverse transcriptase, 0.5 μL RNA sin and 4.5 μL nuclease-free water.A concentration of 7.5 pmoles/μL primer reagent was resuspended with the primer pairs in 50 μL autoclaved deionized water and 0.1X TE buffer (1 mM Tris HCl, pH 8.0 at 25°C; 0.1 mM EDTA, pH 8.0 at 25°C).After the PCR reaction process was completed, the cDNA samples were stored in a refrigerator at -20°C.Expression was quantified using the 2 -ΔΔCt method and the fold-difference of expression levels of genes were calculated and compared in cycle threshold (Ct) values. 21The melting curve was generated immediately after amplification by holding the reaction mixture at 95°C for 60 seconds, and then lowering the temperature to 45°C at a transition rate of 0.1°C/second and maintained for 120 seconds.Then, the samples were heated slowly at a transition rate of 0.05 to 80°C with continuous collection of fluorescence at 640 nm.

Immunocytochemistry
Quantification of cardiomyocyte expression (GATA4 and cTnT) was performed by immunocytochemistry.Assessment of the expression of the differentiation marker of GATA and cTnT in DFAT culture at this stage using the FITC-labelled immunocytochemical method, was carried out on days 7, 14 and 21 after the cells were exposed to differentiation medium.DFAT cells were incubated with a target unmasking fluid (Accurate Chemical & Scientific Corp., Westbury, NY, USA) for 15 minutes using a microwave oven for antigen retrieval.
This was followed by incubation with DAPI (Vector Laboratories).The incubated monolayer cells were fixed with 4% formalin buffer for 15 minutes, then the object glass was washed with PBS and dried.Followed by blocking with 10% PBS for 15 minutes.Immunocytochemistry in the DFAT preparation was intended to observe the expression of cTnT where the cells were then incubated with fluorophore-labelled secondary antibody with F(ab')2-Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 488 (Thermo Fisher Scientific Cat# A48282, RRID: AB_2896345).The staining process was carried out in a dark room because it was sensitive to light, then incubated at 37°C for 1 hour and then observed using an excitation filter of 450-560 nm under a fluorescence microscope (Nikon) at a magnification of 200Â.

Data analysis
We analysed the association between various concentrations of reversine (10, 20 and 40 nM) with the expression of cell stemness biomarkers (Oct4, brachyury, Flk-1 gene expression), and early cardiomyocyte (GATA4) and mature cardiomyocyte (cTnT) markers from the 6 th cell passage.Firstly, we did a normality test using Shapiro-Wilk test.For normally distributed data, we performed a comparison test using Analysis of Variance (ANOVA) with Tukey's post-hoc analysis.For data that was not normally distributed, we performed Kruskal Wallis followed by a Mann-Whitney U test.
Path analysis was carried out using multiple linear regression to determine the pathway mechanism of the influence of the concentration of reversine on the dedifferentiation of DFAT cells and differentiation into mature cardiomyocytes.Statistical tests were performed using IBM SPSS Statistics (RRID:SCR_016479) version 25.0 software.

Characterization of DFAT cells as mesenchymal stem cells
The surface markers used in the DFAT cell characterization process were CD90 and CD105, which are specific markers for mesenchymal stem cells, and CD45 and CD34, which are specific markers for hematopoietic cells.[24] Table 1.Sequences of primer pairs for reverse transcription-quantitative PCR.Analysis of RT-qPCR for gene expression of DFAT (Oct4, brachyury, and Flk-1) DFAT cells were analysed using the RT-qPCR method on days 3, 5, and 7.The mean data from the experiment from the 6 th cell passage is in Table 2.As seen in Table 2, the REV10, REV20, and REV40 groups had a lower mean value of Oct4 gene expression compared with group K. Similarly, for the mean expression of brachyury gene expression, all reversine groups had lower values than the control group.The opposite occurred for Flk-1 gene expression, where reversine groups always have higher mean value than control group, with REV10 and REV40 had the highest value of Flk-1 gene expression.Analysis using the ANOVA test showed that there were significant differences in the mean values of gene expression (Oct4, brachyury, and Flk-1) at each reversine dose (REV10, REV20, and REV40) as can be seen in Table 2.

Primers
The mean value for Oct4 gene expression from the 6 th cell passage showed a decreasing trend with the addition of reversine.There was a statistically significant difference between the concentration of reversine and the gene expression of Oct4 (p = 0.004), with the greatest Oct4 gene expression in the control group and the lowest Oct4 gene expression in the REV40 group, as can be seen in Figure 2.
The mean value for brachyury expression from the 6 th cell passage showed a decreasing trend with the addition of reversine.There was a statistically significant difference between the concentration of reversine and the gene expression of brachyury (p < 0.001), with the greatest brachyury gene expression in the control group and the lowest brachyury gene expression in the REV40 group, as can be seen in Figure 3.
For Flk-1 gene expression from the 6 th cell passage, REV10 increased the mean value of Flk-1 expression from the 3 rd day of observation until the 7 th day of observation, whereas REV20 and REV40 did not show any statistically significant differences (Figure 4).If the REV20 and REV40 variables are omitted, it can be concluded that REV10 significantly increased the mean value of Flk-1 gene expression on the 5 th and 7 th day of observation (p < 0.001).These data were obtained through analysis using independent t-test and paired t-test, with a bar chart in Figure 5.

Immunofluorescence analysis of cardiomyocyte cell markers
The quantity of GATA-4 and cTnT expression from the 6 th cell passage analysed on day 21 showed an abnormal data distribution, so we used the Kruskal-Wallis test for further data processing.The median value of GATA-4 decreased from day 7 until day 21, while the median value of cTnT increased from day 7 until day 21.Kruskal-Wallis test revealed that the expression levels of GATA-4 and cTnT were different between control group, REV10, REV20 and REV40 groups (p < 0.001) as can be seen in Table 3. Figure 6 revealed a trend of decreasing value of GATA4 in all groups between 21 days of observation (p = 0.017).Figure 7 revealed a trend of increasing value of cTnT in all group between 21 days of observation (p = 0.001).

Path analysis
When path analysis was performed on all variables, it was found that at phase 3, the dose of reversine had a significant effect on the quantity of cTnT (p = 0.015) and GATA4 (p = 0.011) expression.Meanwhile, in passage six, the reversine dose only had a significant effect on GATA4 expression (p = 0.004).Meanwhile, the reversine dose had a significant   effect on the quantity of Flk-1 expression (p < 0.001), which indicates how strong the effect of the dose is (a positive sign means it has a positive effect as can be seen in Figure 8.

Expression of cell potency and DFAT "cell stemness"
The ability of DFAT cells to differentiate into multiple derived cells from the three germ layers raises questions about the cell stemness properties of DFAT cells.6][27][28][29][30] DFAT cells show the expression of several cell potency such as Oct4, Sox2, Nanog, Rex-1 31 SOX9, ACAN 32 and Flk-1. 33In this study, DFAT cells were exposed to different doses of reversine with the assumption that the reversine would affect the potency of DFAT cells.There were three markers analysed in this study, namely Oct4, which represents cell stemness transcription factor (SRTF) at the pluripotent level, brachyury, which represents mesodermal cell transcription factor at the multipotent level, and Flk-1, which is a marker of mesodermal endothelial cells.Meanwhile, the doses of reversine used were 10, 20 and 40 nM.The concentration selection was made based on a preliminary study that had been carried out previously using reversine at the concentration of 10, 20 and 40 nM.Analysis of these three markers showed that reversine, especially at the concentration of 10 nM, was able to increase Flk-1 expression consistently during the three days of observation.Reversine at the concentration of 10 nM was also superior to other doses in increasing the expression of Oct4 and brachyury markers even though it had a lower mean value than the control group in all observation groups.

Expression of Flk-1 as a marker of mesodermal endothelial cells
Flk-1 is one of the markers of mesodermal endothelial cells that are essential in the development of endothelial and other hematopoietic derivatives.The detection of this marker confirms the assumption that DFAT cells undergo dedifferentiation from mature mesenchymal cells into more "younger" cells.Expression of Flk-1 in DFAT cells indicates the potency of these cells to differentiate into cardiomyocytes.This is in line with previous studies using an embryonic stem cell differentiation model in mice, showing that progenitor cells expressing Flk-1 can differentiate into contractile cardiomyocytes, whereas cells that do not express Flk-1 are unable to differentiate into contractile cardiomyocytes.

Expression of brachyury as a carcinoma cell transcription factor
Brachyury is a transcription factor that indicates the ability of mesodermal differentiation.Brachyury expression in DFAT cells notably decreases when compared to AMSC cells. 34In this study, the administration of reversine did not increase the expression of brachyury.Perhaps this indicates the ability of reversine to induce further de-differentiation in DFAT cells not to the mesodermal level.Brachyury is also a specific and sensitive marker for the diagnosis of chordoma and has been shown to play a role in the carcinogenesis and development of several epithelial carcinomas. 35A further question that needs to be investigated is whether the low expression of brachyury is associated with a lower risk of DFAT cell carcinogenesis. 36

Oct4 expression as a marker of cell pluripotency
Oct4 is one of the markers of cell pluripotency that plays a role in the differentiation of the three germ layers.Oct4 expression in this study is lower than in several previous studies. 35This could be due to several reasons, the first being the lack of observation days and the second being that the dose in this study was not able to induce an increase in Oct4 expression.

Effect of reversine concentration to cTnT and GATA4 expression
One of the main objectives in this study was to find out the optimal dose for differentiation of DFAT cells into cardiomyocytes.The results of this study indicate that reversine had a significant effect on the expression of cardiomyocyte cell markers.Among the three doses used, reversine at a dose of 10 nM was consistently able to increase the expression of GATA4 and cTnT markers in two different passage groups.Meanwhile, the other two doses, namely the concentration of 20 nM and 40 nM, showed inferior results when compared with the concentration of 10 nM.This supports the assumption that the dose of reversine is concentration dependent on the differentiation ability of DFAT cells.
If you look at the median values of GATA4 and cTnT in each subgroup, you will see a tendency to decrease the median value of GATA4 and increase the median value of cTnT on each day of observation.This is consistent with the theory that states that GATA4 as a marker of cardiac progenitor cells will decrease when DFAT cells differentiate into cardiomyocytes, which are characterized by the detection of cTnT, so it is assumed that GATA4 and cTnT have a negative association, i.e., the lower GATA4 expression the higher the levels of cTnT.However, a different reality was found in this study, particularly in the cTnT group.[39]  In the cTnT group, inconsistency was seen in each passage group, reversine at a concentration of 10 nM increased the median value on days 7 and 14, but decreased values on day 21 in passage three, while in passage six the median value consistently increased.The same thing happened in the 40 nM reversine group.Meanwhile, at a concentration of 20 nM, the median value at passages three and six seemed to decrease with increasing days of observation.

Limitations
The inconsistencies mentioned above can be caused by several technical factors that were not controlled by the researchers, including cell density, which causes false negative results, the emergence of artifacts during staining, which causes false positive results and less than perfect absorption of antibody/Alexa Fluor due to insufficient membrane recovery time achieved.Although passages three and six are still classified as early passages, there are several things that can cause differences in the quality of each of these passages.Senescence is one of the causes of decreased differentiation ability of DFAT cells.It can be concluded that the differentiation ability decreases as cells age.

Conclusions
Reversine could increase the expression of Flk-1, yet it was unable to stimulate the expression of Oct4 and brachyury related to cell stemness.An optimal concentration of 10 nM reversine may have the greatest effect on enhancing the differentiation of DFAT cells into mature cardiomyocytes, as can be seen by higher cTnT expression between cells.

Data availability
Underlying data Figshare: Raw Data -Effects of Different Concentration of Reversine to Enhance Conversion of Dedifferentiated Fat Cells into Cardiomyocyte.https://doi.org/10.6084/m9.figshare.20000426. 22e project contains the following underlying data: • ICC-BB.sav(immunocytochemistry data) • RTqPCR.savFigshare: Raw Ct values and standard curves for all samples and replicates.https://doi.org/10.6084/m9.figshare.20109680. 23gshare: Raw, unedited, uncropped immunocytochemistry images.https://doi.org/10.6084/m9.figshare.20109629. 24ta are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).dedifferentiated fat cells into mature cardiomyocytes", is interesting and relevant to the field of regenerative medicine.The authors have found that reversine, a purine-derivative, could stimulate the differentiation of DFAT cells into mature cardiomyocytes, as indicated by the higher expression of cardiac troponin T, a specific marker of cardiac muscle cells.Although the study is interesting, it has many imprecisions and linguistic flaws leading to ambiguities.

Title:
Rephrase slightly ("Effects of different concentrations of reversine on the conversion of dedifferentiated fat cells into mature cardiomyocytes").

Introduction:
Authors should cite the review article ( Mitrečić D.et.al., 2022 Authors must rephrase the following sentence as it is unclear: "... a higher percentage of the expressed quantity of the mesenchymal stem cell marker known as dedifferentiated fat (DFAT) cells".It should be either "higher quantity" or "higher percentage" of expressed markers.And markers ARE NOT dedifferentiated cells.

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Delete "more" in the sentence "...the dedifferentiation of unipotent myoblast cells to progenitor cells, which are more multipotent".In the next sentence delete "stem".The sentence should read: "Reversine as an inductor of cell dedifferentiation..." ○ "Aims" and "objectives" represent a redundancy.The two paragraphs should be merged, reworded and shortened.

M&M:
It is unclear what was the number of patients whose adipose tissues were used for cell culturing.
○ All units should be very carefully re-checked.For instance "A total of 30-50 L was then transferred" or "A total of 2 L reagent was added to the PCR tube, then 3 L RNA sample was added to the same PCR tube"-µ is missing; also "5.6 microns carrier RNA and 560 micros AVL buffer"-this is incorrect, etc.

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Additionally, authors say 30-50 microliters was transferred into 6-well plates.It changes a lot if there was 20 microliters in one and 50 microliters in another well.Correct this.

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What was done with DFAT after 15 minutes of incubation with reversine?

Results:
Under the subsection "Analysis of RT-qPCR for potential markers of DFAT", there are some discrepancies.Authors say that Oct and Brachyury had lower average values than controls, and that for Flk it was the opposite, when even Flk had lower values compared to controls.Either correct this statement or the table.

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Maybe authors should merge this and the following subsection (as it also deals with relative expressions of Oct, brachyury and Flk genes) and harmonize them.

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The word "gene" is missing after Oct, brachyury, Flk (it should be Oct4 gene expression, etc.) .caption should be corrected ("Graph showing that Oct4 expression is highest in the control group, and decreased in the REV10, REV20 and REV40 groups"), because for REV10, on day 7, there is an increase.

Discussion:
Rather use the word "potency" instead of potential in this case, and correct "stem cell-ness" into ambiguities.

Title:
Rephrase slightly ("Effects of different concentrations of reversine on the conversion of dedifferentiated fat cells into mature cardiomyocytes").
Author response: thank you for your suggestion.We rephrased the title with "Reversine enhances the conversion of dedifferentiated fat cells into mature cardiomyocytes"

Introduction:
Authors should cite the review article ( Mitrečić D.et Authors must rephrase the following sentence as it is unclear: "... a higher percentage of the expressed quantity of the mesenchymal stem cell marker known as dedifferentiated fat (DFAT) cells".It should be either "higher quantity" or "higher percentage" of expressed markers.And markers ARE NOT dedifferentiated cells.

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Author response: thank you for your feedback.We rephrased it with "… with a higher quantity of dedifferentiated fat (DFAT) cells" Delete "more" in the sentence "...the dedifferentiation of unipotent myoblast cells to progenitor cells, which are more multipotent".In the next sentence delete "stem".The sentence should read: "Reversine as an inductor of cell dedifferentiation..." minutes, and then further induced under lineage-specific inducing condition in differentiation medium.Cells grown on coverslips were fixed with 4% paraformaldehyde.Then, we performed RT-qPCR for Oct4, brachyury and Flk-1 on days 7, 14 and 21 after the cells were exposed to differentiation medium.We performed quantification of dedifferentiating mature adipocytes and cardiomyocyte expression (GATA4 and cTnT) were performed by immunocytochemistry.

Results:
Under the subsection "Analysis of RT-qPCR for potential markers of DFAT", there are some discrepancies.Authors say that Oct and Brachyury had lower average values than controls, and that for Flk it was the opposite, when even Flk had lower values compared to controls.Either correct this statement or the table.

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Author response: thank you for your careful observation and feedback.We apologize for the misunderstanding.We have corrected the table.The true statemet is reversine groups always have higher mean value of Flk than control group, with REV40 had the highest value of Flk-1.
Maybe authors should merge this and the following subsection (as it also deals with relative expressions of Oct, brachyury and Flk genes) and harmonize them.

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Author response: thank you for your suggestion, we merged both and deleted the following subsections.
The word "gene" is missing after Oct, brachyury, Flk (it should be Oct4 gene expression, etc.)

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Author response: thank you for your suggestion, we added "gene expression" The standard deviation bars are missing on the histograms ○ Author response: thank you for your consideration and attention to this matter.We added the standard deviation bars on the histograms.It is unclear which cell passage (3 rd or 6 th ) treatments are given in the figures.

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Author response: we used the 6 th cell passage treatments for data analysis and figures.We will explain it in the manuscript.It is not very clear what do the p values above the histogram actually connect.

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Author response: thank you for your consideration and attention to this matter.We try to explain the p values clearer, as a comparison between each group of intervention, not between day. Figure 2. caption should be corrected ("Graph showing that Oct4 expression is highest in the control group, and decreased in the REV10, REV20 and REV40 groups"), because for REV10, on day 7, there is an increase.Author response: thank you for your suggestion, we have corrected the Figure 2 caption with "except on day 7".

Discussion:
Rather use the word "potency" instead of potential in this case, and correct "stem cell-ness" into "cell stemness".Author response: thank you for your suggestion, we replace the word "potential" with "potency" and "stem cell-ness" with "cell stemness" The expression of Oct in this paper and the paper #34 is not really comparable, because the experimental settings are different.
Reviewer Expertise: Regenerative Medicine, gene/cell therapy, Muscular Dystrophy, Molecular and cell biology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
possible to observe a trend in the gene expression for the other 2 concentration REV10 and REV20 is observed a random expression during the time course.has the author an explanation for this result?If yes it could be important to be added in the manuscript.
3-Result in figure 3 is very clear.It well known that downregulation of brachury genes results in down regulation of different mesenchymal marker (e.g.SMA,Vimentin, Desmin) did the author analysis.the expression of this gene?Showing the regulation also of these downstream genes could strength this data and the paper.
4-How can the author correlate the increase of expression of FLK-1 after a treatment with REV10 with the result obtained for Oct4 gene in figure 1 where the expression was affected only after treatment with REV40?
5-The level of expressions of GATA4 and cTnT protein are essential to demonstrate the fully differentiation of stem-cell in cardiomyocyte.To strengthen his result the author has to show the immunofluorescence, to observe also if the cells have also the typical cardiac phenotype (expression of sarcomere, and the shape of the cells).
Author wrote a paragraph about limitations, could be useful for the reader if these limitations are indicated in the results.

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?Partly

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate?No Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Partly
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Regenerative Medicine, gene/cell therapy, Muscular Dystrophy, Molecular and cell biology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
5-The level of expressions of GATA4 and cTnT protein are essential to demonstrate the fully differentiation of stem-cell in cardiomyocyte.To strengthen his result the author has to show the immunofluorescence, to observe also if the cells have also the typical cardiac phenotype (expression of sarcomere, and the shape of the cells).
Answer: Thank you for your suggestion, authors have added Figure 9 (Immunofluorescence of GATA4 in the cell culture) and Figure 10 (Immunofluorescence of cTnT in the cell culture) to support the results.

Competing Interests:
No competing interest to be disclosed The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com and 40 nM), and performed RNA extraction and cDNA synthesis.Next, we used a ; R, reverse; Oct4, Octamer-binding transcription factor 4; Flk-1, Fetal liver kinase 1.

Figure 2 .
Figure 2. Graph showing that Oct4 gene expression is highest in the control group, and decreased in the REV10, REV20 and REV40 groups, except on day 7. Oct4, Octamer-binding transcription factor 4; REV, reversine.

Figure 3 .
Figure 3. Graph showing that brachyury gene expression is highest in the control group, and decreased in the REV10, REV20 and REV40 groups.REV, reversine.

Figure 7 .
Figure 7. Graph showing the increasing expression of cTnT in all groups over 21 days of observation.cTnT, Cardiac troponin T; REV, reversine.

Figure 9 .
Figure 9. Immunofluorescence of GATA4 in the cell culture.

Figure 10 .
Figure 10.Immunofluorescence of cTnT in the cell culture.

○○Figure 2
Figure2.caption should be corrected ("Graph showing that Oct4 expression is highest in the control group, and decreased in the REV10, REV20 and REV40 groups"), because for REV10, on day 7, there is an increase.Discussion: Rather use the word "potency" instead of potential in this case, and correct "stem cell-ness" into

Table 2 .
Mean value and ANOVA for Oct4, Brachyury, and Flk-1 gene expression based on the concentration of reversine.
.al., 2022 [Ref 1])Author response: thank you for your suggestion.We cited the article you've suggested in the reference number 7. ○