Fibroblast growth factor-6 enhances and CDK 2 MAKT expression in microvesicles derived from human stem cells extracted from exfoliated deciduous teeth

Stem cells from human exfoliated deciduous teeth (SHEDs) Background: 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 and gene expression in SHED-derived CDK2 MATK microvesicles (MVs). SHEDs were cultured from deciduous teeth pulp. SHEDs were divided into two groups: the control group and test Methods: groups, with and without FGF-6 supplementation, respectively. After the third passage, SHED proliferation was assessed by MTT assay. MVs were purified and and gene expression was assessed by real time CDK2 MATK polymerase chain reaction. SHEDs were identified by their positivity for CD90 and CD73, and negativity for CD45 and CD34. SHEDs proliferation in the test group was significantly higher than Results: in the control group (P<0.001). mRNA from SHED-derived MVs from the test group exhibited a markedly elevated expression of and , CDK2 MAKT (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. and genes in SHED-derived MVs can be used Conclusions: CDK2 MAKT 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 evaluate SHED proliferation upon supplementation with FGF-6 in comparison with SHEDs in regular culture medium without FGF-6 supplementation; it also aimed to assess the effect of FGF-6 supplementation on CDK2 and MATK gene expression in purified proliferated stem cell MVs.

Sample collection
A total of 28 deciduous teeth indicated for extraction were collected from 25 patients at the Pediatric Dentistry Department in Faculty of Dental Medicine, Cairo University. Patient age ranged from 7 to 12 years. Collection was done at the pediatric clinic over 3 days, we looked for deciduous teeth indicated for extraction due to their natural shedding time in order to make room for their permanent successors, so no ethical concerns would arise. Deciduous tooth collection was conducted after obtainment of the guardians' written informed consent at Pediatric Dentistry Department in the Faculty of Dental Medicine Cairo University, with the approval of the Ethics Committee of the Faculty of Oral and Dental Medicine, Cairo University. Subjects were identified by their treating physician, following which we contacted the guardians of the subjects for consent to use the extracted teeth. Stem cell propagation (at the Medical Biochemistry Department in the Faculty of Medicine Cairo University) was performed in accordance with recommendations and with the approval of the Ethics Committee of the Faculty of Oral and Dental Medicine, Cairo University.
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

Amendments from Version 2
In the new version of this article we have edited the title and abstract to say "MATK" instead of "MAKT", rephrased the conclusion and corrected repeated terminology mistakes. We have also submitted the article for language revision.

See referee reports
REVISED addition, quantification of several expressed MSCs markers was conducted using flow cytometry analysis. Adherent cells were trypsinized and subjected to centrifugation to form cell pellet. Next, 1×10 5 cells were incubated with 10 µl monoclonal CD90 PE (catalog number FAB2067A; R&D Systems), CD73PE (catalogue number FAB5795P; R&D systems) CD34 PE (catalogue number FAB72271P; R&D Systems) and CD45 PE (catalog number DAB1430P; R&D Systems) antibodies, at 4°C in the dark. Same species isotypes served as a negative control, Mouse IgG1 PE conjugated antibody (catalog number IC002P; R&D Systems). 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 2000g for 20 min to remove debris, cell-free supernatants were centrifuged at 100,000g 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 .

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 was significantly higher (P<0.001) in comparison with that of the control group (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) and MATK (P=0.005) 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. The correlation coefficients of CDK2 and MATK expression to cell viability were positive for both genes in the test group (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 .
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 .
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 .
MVs were isolated from the proliferated SHEDs for three reasons. The first reason was to assess the two groups of proliferated SHEDs (i.e. the control group and the group supplemented with FGF-6) via the expression of CDK2 and MATK. The second reason was to explore the MVs RNA since the physiological functions of MVs in many tissues remain mostly unknown; on the other hand, their potential roles in pathological settings have been studied largely in the fields of oncology and immunology. The third reason was that the MVs have great potential for use in research 23 . It was demonstrated that the 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 .
In this study, the 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 24 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 25 .
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
SHED proliferation was significantly higher in culture medium with FGF-6 present than in culture medium with no FGF-6; there was a corresponding increase in positive expression of CDK2 and MATK mRNA from extracted MVs. We recommend further experimentation with microvesicles derived from stem cells to test their effect on differentiation of nerve,bone, cartilage and other tissues.

Grant information
The author(s) declared that no grants were involved in supporting this work.
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.
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