Fibroblast growth factor-6 enhances CDK2 and MATK expression in microvesicles derived from human stem cells extracted from exfoliated deciduous teeth

Background: Stem cells from human exfoliated deciduous teeth (SHEDs) are considered one of the most convenient sources of adult stem cells. This study aimed to examine the effect of fibroblast growth factor 6 (FGF-6) on SHEDs and evaluate CDK2 and MATK gene expression in SHED-derived microvesicles (MVs). SHEDs were cultured from deciduous teeth pulp. Methods: SHEDs were divided into two groups: the control group and test groups, with and without FGF-6 supplementation, respectively. After the third passage, SHED proliferation was assessed by MTT assay. MVs were purified and CDK2 and MATK gene expression was assessed by real-time polymerase chain reaction. SHEDs were identified by their positivity for CD90 and CD73, and negativity for CD45 and CD34. Results: SHEDs proliferation in the test group was significantly higher than in the control group (P<0.001). mRNA from SHED-derived MVs from the test group exhibited a markedly elevated expression of CDK2 and MATK, (P<0.002 and P<0.005, respectively) in comparison with those of the control group. FGF-6 enhanced the proliferation of SHEDs. Proliferation enhancement is favorable for the production of a large number of stem cells, which will then be beneficial for cell-based therapies. Conclusions: CDK2 and MATK genes in SHED-derived MVs can be used as molecular biomarkers for SHED proliferation.


Introduction
Stem cells from human exfoliated deciduous teeth (SHEDs) are a type of adult stem cell acquired from the dental pulp of human exfoliated deciduous teeth. SHEDs stand out from other types of adult stem cells since they possess a remarkable growth and proliferation rate, providing an adequate stem cell source for any prospective clinical or laboratory use. The natural exfoliation of deciduous teeth provides a good chance to procure and isolate SHEDs without effort or complications, and with little or no trauma [1][2][3] .
Fibroblast growth factors (FGFs) are a family of secreted cytokine proteins that have a role in the regulation and direction of numerous cellular processes, including proliferation, differentiation, migration or metabolism 4 . FGF-6 is part of the FGF-4 subfamily of canonical FGFs. Like the other members of FGF-4 subfamily, FGF-6 is a secreted protein with a cleavable N-terminal signal peptide that binds and activates FGF receptors as an extracellular mediator 4 . Despite the fact that FGF-6 expression is almost completely limited to myogenesis 5 , it was found to exert a proliferating effect on human osteoblasts under specific conditions 6 .
Recently, microvesicles (MVs) have been identified as an approach deployed by stem cells as a mean of mediating intercellular interactions 7 . These phospholipid membrane-bound MVs partake in intercellular interactions, such as proliferation, differentiation and gene expression alteration, via their content of mRNA, miRNA and transfer proteins 7,8 .
Human liver stem cell-derived MVs were found to have a role in hepatic regeneration, since they transfer proteins and mRNAs associated with the control of transcription, translation, proliferation, and apoptosis to hepatocytes 9 . Cyclin-dependent kinase 2 (CDK2) and megakaryocyte-associated tyrosine kinase (MATK) genes are associated with cellular proliferation as they were found in the mRNA of purified MVs at the time of microarray analysis and reverse transcription-quantitative PCR (RT-qPCR) 9 .
CDK2 is the catalytic subunit of the cyclin-dependent protein kinase complex, which controls advancement through the cell cycle via its involvement in the G 1 to S phase transition 10,11 . MATK has been identified by Avraham et al. as an intracellular tyrosine kinase that participates in the proliferation and survival of megakaryocyte progenitors 12 . Furthermore, Findings by Herrera et al. demonstrated that MATK conveyed by MVs was one of the genes responsible of liver stem cell proliferation 9 .
The current study was performed to use SHEDs derived microvesicles as biomarker for cellular proliferation after FGF-6 supplementation by assessing the CDK2 and MATK gene expression in microvesicles' mRNA. Deciduous tooth surfaces were washed several times with Dulbecco's PBS (Biowest, USA). Dental pulp was extracted delicately from teeth using a sterile endodontic barbed broach and placed in falcon tube containing PBS (Biowest, USA).

SHED culture and characterization
SHEDs culture and characterization were done after taking established procedures into account 13 . A total of 3 mg collagenase type II (Sigma Aldrich, USA) was dissolved in PBS to digest the extracted dental pulp tissues for 1 h at 37°C in a 5% CO 2 incubator and shaken well at 10 min intervals until the tissues were fully digested. The samples were strained using a cell strainer (40 µm nylon PP) (Bio Basic, Inc., Canada) to remove tissue debris and then centrifuged for 10 min at 3000 rpm at 5°C to obtain pellets of isolated cells. The supernatant fluid was discarded and cell suspension was obtained by pipetting cells in RPMI 1640 culture medium (Biowest, USA). Next, the isolated cell pellets were seeded in 75 cm 3 tissue culture flasks for cell culture propagation. Culture medium (RPMI 1640) (was supplemented with 1% Pen/Strep solution (Lonza, USA) and 10% fetal bovine serum (FBS) (Lonza, USA) were supplemented to the culture media to achieve cell propagation at 37°C in humidified CO 2 incubator for 7-10 days, with medium changes every 3 days.
Cells were identified as being mesenchymal stem cells (MSCs) by their morphology and adherence to the plastic flask. In addition, quantification of several expressed MSCs markers . After a 20 min incubation, 2 ml PBS containing 2% FBS was added to a tube of monoclonal treated cells. The mixtures were then centrifuged for 5 min at 2500 rpm, followed by discarding the supernatant and re-suspending cells in 500 µl PBS containing 2% FBS. Cell analysis was performed using a CYTOMICS FC 500 Flow Cytometer and analyzed using CXP Software version 2.2.

SHEDs proliferation process and passaging
Passaging of SHEDs was done according to established protocols 14 , with modifications for this experiment. Sub-culturing and passaging was done when adherent cells primary culture (passage zero) have reached 80% confluence. 10 3 -10 5 cells were seeded into 24-well plates prior to grouping and subsequent passaging till reached third passages. Seeded cells were divided into two groups: control group (SHEDs untreated with FGF-6) and test group (SHEDs treated with FGF6). FGF-6 was added at concentration 20 ng/ml for test group.
Cell viability MTT reagent, supplied ready for use after the third passage of the SHEDs, was obtained from Tacs Trevigen (Gaithersburg, USA). For the cell viability assay, the two cell groups were seeded in three 96-well tissue culture plates each, at 10 3 cells/ml per well. The MTT reagent was added and the plate was incubated in the dark for 2-4 h. Detergent reagent (catalog number # 4890-25-02, TACS) was added to each well to solubilize formazan dye prior to absorbance measurement. The absorbance in each well was measured at a range from 490 to 630 nm using an enzyme-linked immunosorbent assay plate reader (Stat Fax 2200, Awareness Technologies, Florida, USA) 15 .

MV isolation
MVs were obtained from supernatants of third-passage MSCs (5×10 6 cells/ml) cultured in RPMI-1640 deprived of FBS and supplemented with 0.5% of bovine serum albumin (BSA) (Sigma Aldrich, USA). After centrifugation at 2000 g for 20 min to remove debris, cell-free supernatants were centrifuged at 100,000 g for 1 h at 4°C, washed in serum-free medium 199 containing 25 mM HEPES (Sigma) and submitted to a second ultracentrifugation under the same conditions 16 . MVs were then prepared for electron microscopy characterization. Briefly MVs were diluted in 145 µL PBS containing 0.2% paraformaldehyde (w/v). 10 µl was administered to a formvar-carbon-coated 300 mesh grid (Electron Microscopy Sciences, Hatfield, USA) for 7 min, followed by staining with 1.75% uranyl acetate (w/v). Samples were left to dry at room temperature for 2 h and imaged by transmission electron microscopy (TEM) (CM-10, Philips, Eindhoven, The Netherlands) at 100 kV afterwards 17 .

Gene expression profile
Total RNA was isolated from MVs using an RNA purification kit (Gene JET, Kit, #K0731, Thermo Fisher Scientific, Inc.). RNA quantification using RT-qPCR was achieved using a one-step reaction (SensiFAST™ SYBR® Hi-ROX One-Step Kit, catalog no. PI-50217 V; Bioline, UK). Sequence-specific primers (Bio Basic, USA) for the studied target genes (CDK2 and MATK) and reference housekeeping gene (β-actin) were used. The prepared reaction mix samples were applied in real time PCR (StepOne Applied Biosystem, Foster city, USA). The cDNA was subsequently amplified using a SYBRGreen I PCR Master kit (Fermentas) in a 48-well plate as follows: 10 min at 95°C for enzyme activation, followed by 40 cycles of 15 s at 95°C, 20 s at 60°C and 30 s at 72°C for the amplification step. Changes in the expression of each target were normalized relative to the mean Cq values of β-actin as housekeeping gene by the 2 −∆∆Cq method. We used 1 µM of both primers specific for each target gene. Primers sequences were as follows:

Statistical analysis
Data were coded and entered using SPSS version 23. Data are presented as the median and interquartile range for quantitative data Comparisons between quantitative variables were done using the non-parametric Mann-Whitney test. Correlations between quantitative variables were done using Spearman's correlation coefficient. P-values less than 0.05 were considered as statistically significant.

SHED characterization
Cultured SHEDs exhibited fusiform fibroblast like appearance for both groups. During culture and passaging, SHEDs in the test group proliferated more than SHEDs in the control group ( Figure 1). Flow cytometric analysis for SHEDs was negative for CD34 and CD45 and positive for CD90 and CD73 ( Figure 2A).

Cell viability
The viability of the cells in the test group (n=17) was significantly higher (P<0.001) in comparison with that of the control group (n=17) ( (n=17) ( ( Table 1).
TEM TEM detected MVs purified from SHED after ultracentrifugation ( Figure 2B). MVs were characterized by their size (500 nm), as detected by TEM.

RT-qPCR
Purified MVs demonstrated a significant positive expression intensity of CDK2 (P=0.002) (n=17), and MATK (P=0.005) (n=17)     in the test group in relation with the control group. A box plot (Figure 3) shows that expression of CDK2 and MATK is higher in the test group than the control group, as they display a higher interquartile range (IQR) and medium. Expression of CDK2 is positively correlated with cell proliferation in the test group (P=0.010) (r=0.480). Expression of MATK is positively correlated with cell proliferation in the test group (P=0.031) (r=0.409) (Figure 4).

Discussion
We performed this experiment to deal with difficulties sourcing stem cells and a lack of sufficient stem cells for reliable tissue formation. The study focused on stem cells isolated from human deciduous teeth (SHEDs) and tissue-inducing substances, which in this case is FGF-6. The reason we chose SHEDs for the isolated stem cells is that they present an opportune source of adult stem cells; the deciduous teeth are naturally exfoliating so there are no ethical problems surrounding their use, and the isolation of cells is simple, painless, convenient and time-efficient 18 .
.00 There are several criteria for SHED identification; we identified stem cells by their morphology under the inverted microscope, as they appeared as fibroblast-like cells. Another feature exhibited was that they have a plastic adherence feature under our normal culture conditions 1 . SHEDs were also identified as ectomesenchymal stem cells through the quantification of several expressed mesenchymal stem cell markers using flow cytometry; they were shown to be positive for CD90 and CD73, and negative for CD45 and CD34 19 .

SHEDs
In this case, the stem cells are quiescent 20 , unlike progenitor cells, meaning growth factor treatment is required to produce a large amount of cells. In our experiment, FGF-6 was chosen as it has, to our knowledge, never used on SHEDs before, and would avoid the conflicting reports of the effects of bFGF on SHEDs 21,22 .
SHEDs group with added FGF-6 demonstrated increased cells vitality and number in comparison with the control group. It was evidenced by the MTT assay results and increased expression of both CDK2 and MATK genes present in RNA of microvesicles we purified from SHEDs. We considered microvesicles as a good indicator or a biomarker for cellular proliferation of stem cells in general and SHEDs in particular. Other studies highlighted the importance of microvesicles 'cargo: They can be used as a biomarkers of tumor cells proliferation and progression, cardiometabolic disorders, immunologic diseases, and also cell-derived MVs are found to be able to change phenotypes of different cells to become stem cells via epigenetic reprograming or infectious particle transfer 23-27 . CDK2 gene was used to obverse SHEDs proliferation since it has been used to monitor proliferation in many types of stem cells, such as neural progenitor stem cells 28 and liver stem cells 9 . CDK2 also encodes a serine/threonine protein kinase family member, with receptors in this family having a role in the regulation of cell proliferation, programmed cell death, cell differentiation, and embryonic development 29 .
Megakaryocyte-associated tyrosine kinase is the enzyme which is encoded by MATK in humans. This enzyme possesses a similar amino acid sequence to tyrosine-protein kinase CSK. It was chosen for our experiment as it is not frequently used for the assessment of SHED proliferation, to evaluate whether this ambiguous gene can be studied in further research to assess the proliferation rate of SHEDs and other types of stem cells it is known to be capable of phosphorylating and inactivating Src family kinases, and may inhibit T-cell proliferation 12 .

Conclusion
The present study showed an increased expression of CDK2 and MATK genes present in RNA of microvesicles derived from SHEDs after FGF -6 supplementation. Thus, MVs derived from SHEDs can be used as a biomarker for cellular proliferation.

Recommendations
We recommend that in order to properly verify SHEDs as mesenchymal stem cells not dental pulp fibroblast: STRO1, NANOG, SOX family, OCT4 genes needs to be identified along the experimental process, and multipotential differentiation test should be carried out.
Additionally, future research should take into consideration the differentiation potential of these stem cells derived microvesicles and how they compare to the SHEDs differentiation potential before and after growth factor application, utilizing and comparing different isolation protocols for microvesicles, and testing more cargo genes and CDs.
Furthermore, a future protocol should be formulated and tested to utilize microvesicles derived from stem cells as a biomarker for measuring stem cells' proliferation, via identifying and measuring expression of the genes associated with proliferation, as well as identifying the genes associated with stem cells' differentiation. That future protocol will be applicable in clinical research that involve stem cells, such as hematopoietic stem cell transplantation, as well as preclinical tissue regeneration experiments such as bone, periodontal, neural regeneration, and regenerative endodontics.

Data availability
Underlying data

Thanaphum Osathanon
Center of Excellence for Regenerative Dentistry, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand In the revised version, the authors did not respond to all issues raised by the reviewer. Please respond to or rebut each item separately.
According to Dominici (2006) , mesenchymal stem cells can be characterized by 1) plastic et al. adherence properties, 2) surface marker expression, and 3) multipotential differentiation ability. This article lacks of mutipotential differentiation test. Hence, the claim of these cells as stem cells isolated from human exfoliated deciduous teeth was not accurate. Indeed, these cells may be dental pulp fibroblast cells. If authors cannot provide the evidences of MSCs in this article or citation from authors previous publication, these cells can be call dental pulp cells.
Authors did not mention which time points that they evaluated for cell viability and proliferation. To determine cell proliferation, at least 2 time points are required and the second time points should be longer than the normal doubling time of these cells.
Please provide more detail on those reagent used in the study. The catalog number as well as company is crucial information for others to replicate these finding. The main reagents (for example FGF6) were lacking this information.
In TEM results, it seems that there were a lot of contaminated substance in MV collection. Would this effect the evaluation of gene expression since the RNA from the cells might also contaminated with MVs RNA?
In PCR results, it seems that there was no statistically significant difference. This might be due to the different baseline expression among donors. Plots demonstrated the individual data could help to visualize the trend between control and treatment in each donors.  Overall, the results seems preliminary and required further improvement to strongly support the conclusion claimed by authors. 1.

Department of Endodontics, School of Dentistry, Isfahan University of Medical Sciences, Isfahan, Iran
This study is about evaluation of the effect of FGF-6 on SHEDs and assessment of CDK2 and MATK gene expression, in which the authors reported that CDK2 and MATK genes in SHED-derived can be used as molecular biomarkers for proliferation.
Add mean and SD of patients and add female to male ratio or male to female ratio.
Please provide approval code or public link.
For characterization only flow cytometry is not enough, RT-PCR on genes that show mesenchymal stem cells have been isolated would be interested, e.g. STRO1, NANOG, SOX family, OCT4. Or differentiation to osteoblast, chondroblast and adipocytes.
Please add a paragraph that show how your results would be helpful to clinical research.
Increase the quality of figures.
In Figure 3 for CDK2 there are two outliers data. Figure 4 can be removed. Authors can report the correlation as follows in the results section: Expression of CDK2 is positively correlated with cell proliferation in the test group (r=?, P_value:?)

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 I would like to thank you very much for your review. I truly appreciate your time and effort. We did the following modifications to the article as requested, The Recommendation section was created to state any future experiment carried out should include a multipotential differentiation test and more genes testing to correctly identify adult stem cells to avoid confusion with dental pulp fibroblasts. Additionally, a paragraph about potential use in clinical research has been added. In the correlation section, we added (r) and (P) values. We added sample size number (n) in the cells viability section. We rectified the mistake of adding the GAPDH sequence instead of a beta-actin sequence, by adding beta-actin sequence and accession number. We also recommend in the future to take into consideration male to female ratio and standard deviation during sample collection. We tried to increase the quality of the figures but unfortunately, we could not, because of the status of equipment during the experiment. In future experiments, we will certainly take that into consideration. The ethics approval has been provided to F1000Research editors before publishing.
Unfortunately, the funding for the experiment was very limited, so I could not do the multipotential differentiation test or test the presence of STRO1, NANOG, SOX family, OCT4 genes. In the future, we will apply for a much larger fund to conduct the experiments more properly.

My best regards
No competing interest Competing Interests: 1.
The present article described the effect of FGF6 on RNA contents of microvesicles. In addition, the correlation of detected RNA on cell proliferation was described. There are many issues required to address in order to improve scientific content of this article.
According to Dominici (2006) , mesenchymal stem cells can be characterized by 1) plastic et al. adherence properties, 2) surface marker expression, and 3) multipotential differentiation ability. This article lacks of mutipotential differentiation test. Hence, the claim of these cells as stem cells isolated from human exfoliated deciduous teeth was not accurate. Indeed, these cells may be dental pulp fibroblast cells.
Authors did not mention which time points that they evaluated for cell viability and proliferation. To determine cell proliferation, at least 2 time points are required and the second time points should be longer than the normal doubling time of these cells.
Please provide more detail on those reagent used in the study. The catalog number as well as company is crucial information for others to replicate these finding. The main reagents (for example FGF6) were lacking this information.
How many cells were seeded in the tissue culture plates and treated with FGF6 for further cell viability test and MV isolation? Authors mentioned 10 -10 cells and this is a wide range. Different cell density could reflect the different results obtained.
In PCR, authors indicated the use of beta-actin as reference gene but provided GAPDH sequence.
In TEM results, it seems that there were a lot of contaminated substance in MV collection. Would this effect the evaluation of gene expression since the RNA from the cells might also contaminated with MVs RNA?
In PCR results, it seems that there was no statistically significant difference. This might be due to the different baseline expression among donors. Plots demonstrated the individual data could help to visualize the trend between control and treatment in each donors.
What is the r for correlation analysis? Please indicate the statistical number.
Please indicate n in each experiment.
Overall, the results seems preliminary and required further improvement to strongly support the conclusion claimed by authors.