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

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 account¹³. 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% CO₂ 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 cm³ 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 CO₂ 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 was conducted using flow cytometry analysis. Adherent cells were trypsinized and subjected to centrifugation to form cell pellet. Next, 1×10⁵ 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 protocols¹⁴, with modifications for this experiment. Sub-culturing and passaging was done when adherent cells primary culture (passage zero) have reached 80% confluence. 10³–10⁵ 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 10³ 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 (5×10⁶ 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 conditions¹⁶. 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 afterwards¹⁷.

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: CDK2 sense, 5'-AATCCGCCTGGACACTGAGA-3' and antisense, 5'-CCAGCAGCTTGACAATATTAGGA-3' (Genbank accession number XM011537732.1); MATK sense, 5'-CCGCGACGTCATCCACTAC-3' and antisense, 5'-TTGTAATGCTCCACCATGTCCAT-3' (Genbank accession number AH006874.3); and GAPDH sense, 5'-GGCGGCACCACCATGTACCCT-3' and antisense, 5'-AGGGGCCGGACTCGTCATACT-3' (Genbank accession number AAG01996.1).

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

The SHEDs in culture were identified morphologically by their fusiform fibroblast like form for both groups. SHEDs in the test group exhibited an increase in proliferation during culture and passaging, when compared with the control group (Figure 1). Flow cytometric analysis SHEDs were identified as they were negative for CD34 and CD45 and positive for CD90 and CD73 (Figure 2A).

Figure 1. Isolation, morphological observation of stem cells from human exfoliated deciduous teeth through phase contrast microscopy.

(A) Passage one shows stem cells with spindle-like morphology as grow from human exfoliated deciduous teeth pulp in few number. (B and C) Passages two (B) and three (C) show an increase in number of stem cells with spindle-like morphology. Isolation, morphological observation of stem cells from human exfoliated deciduous teeth in the test group through phase contrast microscopy. (D) Passage one shows a marked increase in number and confluency of stem cells with spindle-like morphology in comparison with control group in passage one. (E and F) Passages two (E) and three (F) show a pronounced, confluent and expanded SHED with fibroblast like morphology in relation to control groups of second and third passages.

Figure 2. Flow cytometry and transmission electron microscopy.

(A) Flow cytometry analysis of CD90, CD73, CD34 and CD45 for stem cell characterization. (B) Electron microscopy ultrastructure of released microvesicles (black arrow) from the mesenchymal stem cells of dental pulp.

Cell viability

The proliferated SHEDs were assessed using MTT assay. 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).

Figure 3. Box-and-whiskers plot showing number distribution for CDK2 and MATK expression in both test and control groups.

(A and B) Expression of each gene is higher in the FGF-6-supplemented group than in the control group, since higher interquartile range (IQR) and median values are observed.

Figure 4. Assessment of cell proliferation.

(A) Expression of CDK2 is positively correlated with cell proliferation in the test group. (B) Expression of MATK is positively correlated with cell proliferation in the test group.