Introduction

Mesenchymal stem cells (MSCs) are a widely used source of stem cells in both basic research and clinical applications. To date, MSCs have been used in the treatment of more than 50 different diseases, included knee osteoarthritis (Emadedin et al. 2015; Kim et al. 2015), wound healing (Isakson et al. 2015; Li et al. 2015b), autoimmune disease (Munir and McGettrick 2015), bone regeneration (Padial-Molina et al. 2015), femoral head necrosis (Daltro et al. 2015; Wang et al. 2015), foot ulcer (Sener and Albeniz 2015), severe systolic heart failure (Zhao et al. 2015), sequelae of traumatic brain injury (Wang et al. 2013), stroke (Gutierrez-Fernandez et al. 2015), cardiac diseases (Karantalis and Hare 2015), Crohn’s disease (Labidi et al. 2014), intervertebral disc repair (Mochida et al. 2015), diabetes mellitus (Kong et al. 2014; Liu et al. 2014), and graft-versus-host disease (Introna and Rambaldi 2015). Thus far, according to ClinicalTrials.gov (www.clinicaltrials.gov), more than 500 clinical trials have used MSCs in disease treatment. Although many MSC sources have been discovered, such as bone marrow (Phadnis et al. 2011), adipose tissue (Estes et al. 2010), peripheral blood (Kassis et al. 2006), umbilical cord blood (Zhang et al. 2011), banked umbilical cord blood (Phuc et al. 2011), umbilical cord (Cutler et al. 2010; Farias et al. 2011), umbilical cord membrane (Deuse et al. 2011), umbilical cord vein (Santos et al. 2010), Wharton’s jelly of the umbilical cord (Zeddou et al. 2010; Peng et al. 2011), placenta (Semenov et al. 2010; Pilz et al. 2011), decidua basalis (Lu et al. 2011), ligamentum flavum (Chen et al. 2011), amniotic fluid (Gucciardo et al. 2013), amniotic membrane (Chang et al. 2010), and dental pulp (Yalvac et al. 2010), researchers continue to search for new sources.

Gingival gum tissue currently serves as a novel source of MSCs, and gingival stem cells (GSCs) are considered important MSCs for jawbone and teeth regeneration as well as gum disease. While teeth implantation is a commonly used technique for patients with missing teeth, this technique is only performed when the jawbone is responsive to the procedure. Therefore, some studies have attempted to regenerate the jawbone using MSCs from bone marrow before performing implants. However, the major limitation for wide use of GSCs as a source of MSCs is the relatively low number of GSCs that can be isolated from gum tissue.

Ascorbic acid (AA) is a commonly used vitamin in medicine. Previous studies revealed AA as an essential agent in stem cell proliferation as well as triggering pluripotent markers in both adult stem cells (Kim et al. 2014; Wei et al. 2014; Yu et al. 2014; Bae et al. 2015; Li et al. 2015a) and embryonic stem cells or induction of pluripotent stem cells (Esteban and Pei 2012; Gao et al. 2013).

In this study, we investigated the effects of AA as a proliferating agent for GSCs with the potential aim to increase proliferation without changes in GSC stemness.

Materials and Methods

Gum tissue collection.

Gum tissues were collected from gum tissue disease patients. Informed consent was obtained from all patients. The collection was performed in accordance with the ethical standards of the local ethics committee.

GSC isolation and culture.

Gum tissues were washed three times with phosphate-buffered saline (PBS) (Invitrogen-Gibco, Carlsbad, CA) supplemented with antibiotic-antimycotic 1× solution. Samples were cut into 0.5–1 cm3 pieces and suspended in Dulbecco’s modified Eagle’s medium (DMEM)/F12, 10% fetal bovine serum (FBS), 1% antibiotic-antimycotic solution (all from Sigma-Aldrich, St. Louis, MO). The tissue was left undisturbed for 4 d in a 37°C humidified incubator with 5% CO2. Culture medium was replaced every 3 d after this time. The cells were passaged using 0.25% trypsin/EDTA solution (Sigma-Aldrich) when cells reached 80–90% confluence. GSC candidates were subcultured to the fifth passage, and these cells were used for further experiments.

Phenotype analysis.

Primary antibodies against human antigen CD13, CD14, CD34, CD44, CD45, CD73, CD90, and CD105 and histocompatibility antigen DR alpha chain (HLA-DR) were purchased from BD Biosciences (San Jose, CA). MSCs (5 × 105 cells) were re-suspended in 500 μL PBS and stained with fluorescein-isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated primary antibodies for 20 min at room temperature. FITC- or PE-conjugated human IgGs were used as isotype controls at the same concentration as the specific primary antibodies. The fluorescence intensity of the cells was evaluated by flow cytometry (FACSCalibur; BD Biosciences). Data were analyzed using CELLQUEST software (BD Biosciences).

Differentiation assays.

For differentiation into adipogenic cells, GSCs (fifth passage) were plated at 1 × 104 cells/well in 24-well plates. At 70% confluence, the cells were cultured for 21 d in DMEM containing 0.5 mmol/L 3-isobutyl-1-methyl-xanthine, 1 nmol/L dexamethasone, 0.1 mmol/L indo-methacin, and 10% FBS (all from Sigma-Aldrich). Adipogenic differentiation was evaluated by observing lipid droplets in cells under a microscope.

For differentiation into osteogenic cells, GSCs were plated at 1 × 104 cells/well in 24-well plates. At 70% confluence, the cells were cultured for 21 d in DMEM/F12 containing 10% FBS, 10−7 mol/L dexamethasone, 50 μmol/L ascorbic acid-2 phosphate, and 10 mmol/L β-glycerol phosphate (all purchased from Sigma-Aldrich). Osteogenic differentiation was confirmed by Alizarin red staining.

For differentiation into chondrogenic cells, GSCs were induced to differentiate by a commercial medium for chondrogenesis (StemPro Chondrogenesis Differentiation Kit, A10071-01; Life Technologies, Carlsbad, CA). GSCs were differentiated in pellet form according to the manufacturer’s guidelines. After 21 d, the cell pellets were stained with an anti-aggrecan monoclonal antibody (BD Biosciences).

MTT assays.

Cells were grown in culture medium DMEM/F12 supplemented with 10% FBS, 1% antibiotic-antimycotic solution, and with different concentrations of AA (10, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μM). AA stock solution was prepared by diluting 14.45 g L ascorbic acid 2-phosphate in sterile 1 L H2O to get 50 mM solution, filtered, and stored at 4°C. This solution was added into culture medium to get the suitable concentration of AA. The effects of AA on GSC proliferation were evaluated by 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay and xCELLigence assay.

About MTT assay, briefly, 20 μL of MTT (5 g/L; Sigma-Aldrich) was added to each well of 96-well plates, followed by incubation for 4 h and addition of 150 μL of DMSO (Sigma-Aldrich). Plates were then mixed well for 10 min until the crystals dissolved completely. Absorption values (A value) for each well was measured at a wavelength of 490 nm using the microplate reader DTX 880 (Beckman Coulter, GmbH, Krefeld, Germany). An offset value of A and absorption value of the control group reflect GSC proliferation.

xCELLigence assay.

To confirm the effects AA on GSC proliferation from MTT assay, we used another assay, so-called xCELLigence assay. This assay used xCELLigence Real-Time Cell Analyzer equipment (Roche Applied Science, Indianapolis, IN). xCELLigence Real-Time Cell Analyzer was used to evaluate cell proliferation and cytotoxicity based on changes in electric impedance at the surface of the E-plate, a specific plate with electric nodes on the surface allowing measurement of changes in impedance. This method was used to evaluate cell proliferation for adherent cells.

A total of 1 × 103 cells were seeded into each well of a 96-well E-plate in triplicate. The culture plates were placed into the eXCELLigence system and incubated at 37°C with 5% CO2. Cell proliferation was monitored for 240 h with fresh medium changes every third day. The cell doubling time was determined by the software of the eXCELLigence system.

Flow cytometry of marker expression.

To analyze MSC marker expression, cultured GSCs were deattached by trypsin/EDTA and washed twice with cold PBS. Cells were stained with primary antibodies against human antigen CD13, CD14, CD34, CD44, CD45, CD73, CD90, CD105, and HLA-DR and then stained with FITC- or PE-conjugated secondary antibodies. The fluorescence intensity of the cells was evaluated by flow cytometry, and data were analyzed using CELLQUEST software.

To evaluate pluripotent stem cell marker expression, GSCs treated with AA were evaluated for SSEA-3, Sox-2, Oct-3/4, Nanog, and TRA-1-60 expression by flow cytometry. GSCs in untreated medium were used as a control. Anti-SSEA-3, anti-Sox-2, anti-Oct-3/4, anti-Nanog, and anti-TRA-1-60 antibodies were purchased from Santa Cruz Biotechnology, Canada.

Real-time RT-PCR.

To evaluate pluripotent stem cell markers, GSCs were examined for SSEA-3, Sox-2, Oct-3/4, Nanog, and TRA-1-60 messenger RNA (mRNA) expression. GSCs in experimental groups were collected by trypsinization, and mRNA was isolated using the Trizol protocol. Obtained total RNA was used as a template for one-step real-time RT-PCR. Real-time RT-PCR reaction was performed using the SYBR® Green Quantitative RT-qPCR Kit (Sigma-Aldrich).

Tumorigenicity assay.

The tumorigenicity of GSCs was examined in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. All experiments and handling of mice were approved by the Local Ethics Committee of Stem Cell Research and Application, University of Science (Ho Chi Minh City, Vietnam). Each mouse was injected subcutaneously with 5 × 106 GSCs (three mice per group). Tumor formation in mice was followed up for 3 mo.

Statistical methods.

The results are expressed as the mean ± SD. One-way ANOVA and two-tailed tests were used for all statistical analyses and performed with GraphPad Prism software, version 4.0. P values less than 0.05 were considered statistically significant.

Results

Gingival stem cell isolation.

Similar to methods described in previous publications, in this study, we cultured isolated GSCs by tissue expansion culture. Three samples were collected and cultured, and all samples appeared as single cells adhering to the flask surface from day 5 after seeding (Fig. 1a ). However, at this time, there were two kinds of cells appearing: epithelial-like cells and mesenchymal-like cells (Fig. 1b ). After 10 d of cell migration from tissue, tissues were removed and cells were subcultured at a dilution factor of 1:3. The GSC candidates rapidly proliferated after the first subculture. The cell population became homogenous in shape after the fifth subculture with a fibroblast-like shape (Fig. 1c ). These cells could strongly form colony (Fig. 1d–i ).

Figure 1
figure 1

Isolation of GSCs by tissue explant culture. a Cell growth from tissues at day 5. b GSC candidates at first subculture (b) and after five passages (c). GSC candidate form colony after 3 d (d), 5 d (e), 7 d (f), 9 d (g), 12 d (h), and 15 d (i).

Full size image

Gingival stem cell characterization.

GSC candidates at the fifth passage were examined for expression of surface markers for MSC phenotype as well as differentiation potential into adipocytes, osteoblasts, and chondrocytes. The results demonstrated that GSC candidates expressed the MSC-specific marker profile, including positive CD13, CD44, CD73, CD90, and CD105 expression and negative CD14, CD34, CD45, and HLA-DR expression (Fig. 2a–h ).

Figure 2
figure 2

GSCs exhibited the MSC phenotype. GSCs were positive for CD44 (c), CD73 (e), CD90 (f), and CD105 (g) expression and negative for CD14 (a), CD34 (b), CD45 (d), and HLA-DR (h). GSCs successfully differentiated into adipocytes (j, k), osteoblasts (l), and control (i).

Full size image

More importantly, the cells also successfully differentiated into two kinds of mesoderm cells including adipocytes and osteoblasts (Fig. 2j, k, l ). In fact, before differentiation, GSCs exhibited fibroblast morphology (Fig. 2i ), and after differentiation, they showed storage of lipid drops inside the cytoplasm to become adipocytes (Fig. 2j, k ) and calcium matrix accumulation (alizarin red positive) to become osteoblasts (Fig. 2l ).

AA stimulated GSC proliferation in a concentration-dependent manner.

We next examined GSC proliferation in response to various concentrations of AA. Proliferation was enhanced in a concentration-dependent manner; at the lowest concentration of AA (10 μM), AA stimulated GSC proliferation, and proliferation of GSCs gradually increased from 10 to 250 μM AA. At 250 μM, the proliferation rate of GSCs was at maximum levels and significantly higher compared with other concentrations (p < 0.5). At this concentration, the doubling time was clearly reduced from 40 h (in control group) to 32 h (in 250 μM AA). However, at concentrations higher than 250 μM, GSC growth was partially or totally inhibited. At 300 and 350 μM, GSCs slightly inhibited with nonsignificant difference to 250 μM but from 250 to 500 μM, GSCs strongly inhibited the sudden decrease in proliferation rate and extended the doubling time (Figs. 3 and 4).

Figure 3
figure 3

GSC proliferation was evaluated by xCELLigence assay. GSCs were cultured in E-plate and treated with 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, and 500 μM of AA and control (without AA). The GSC proliferation in all groups was recorded by xCELLigence machine (a). The doubling time of GSCs in different groups were calculated (b).

Full size image
Figure 4
figure 4

AA triggered GSC proliferation at 250 μM AA. a, Cell proliferation curve and b, doubling time.

Full size image

These observations were confirmed by cell cycle analysis. The results showed that 10–250 μM AA treatment of GSCs increased the percentage of cells in S and G2/M phases. In particular, treatment with 250 μM AA significantly increased the percentage of cells in G2/M + S phases compared with other groups (Fig. 4) (p < 0.5). At concentrations higher than 250 μM AA, almost all cells were at G0/G1 and the percentage of cells at G0/G1 was significantly increased in response to 500 μM AA (p < 0.5).

AA triggered the expression of pluripotent markers.

After 2 wk of treatment with 250 μM AA, GSCs showed increased expression of pluripotent markers including SSEA-3, Sox-2, Oct-3/4, Nanog, and TRA-1-60 (Fig. 5). Both real-time RT-PCR and flow cytometry showed that these markers were increased in AA-treated GSCs compared with controls. SSEA-3 showed the highest expression, and nearly 100% of treated cells strongly expressed this marker after 2 wk. Expressions of Sox-2, Oct-3/4, Nanog, and TRA-1-60 were 32.41, 45.31, 31.03, and 19.79% higher than controls, respectively (p < 0.5). Expression of SSEA-3 mRNA was approximately 503.33 ± 180.09 times higher than controls, while Sox-2, Oct-3/4, Nanog, and TRA-1-60 mRNA levels were 237.33 ± 97.44, 184.67 ± 52.60, 99.67 ± 18.48, and 88.00 ± 13.53 times higher, respectively, compared with controls (p < 0.5).

Figure 5
figure 5

Expression of pluripotent markers in GSCs treated with AA. Upon treatment with 250 μM AA, GSCs strongly expressed SSEA-3 and highly expressed Sox-2, Oct-3/4, Nanog, and TRA-1-60 as shown by flow cytometry analysis (a) and RT-PCR (b).

Full size image

Despite expression of pluripotent stem cell markers, GSCs maintained the MSC phenotype.

GSCs treated with AA still maintained the MSC phenotype and showed a MSC marker profile that was positive for CD44, CD73, CD90, and CD105 expression and negative for CD14, CD34, CD45, and HLA-DR (Fig. 6a–h ). GSCs also successfully differentiated into adipocytes, osteoblasts, and chondrocytes in the inducing medium. In the adipocyte-inducing medium, GSCs stored lipid drops and these drops gradually grew larger after 1 mo of induction (Fig. 6j, k ). In the osteoblast-inducing medium, cells strongly produced matrix with high calcium that was positive for alizarin red staining (Fig. 6l ). More importantly, although these cells showed upregulation of the pluripotent markers, they could not cause tumors in NOD/SCID mice (Fig. 7).

Figure 6
figure 6

AA-treated GSCs maintained the MSC phenotype. AA-treated GSCs expressed MSC markers such as CD44, CD73, CD90, and CD105 and were negative for CD14, CD34, CD45, and HLA-DR (a–h). GSCs treated with AA also successfully differentiated into adipocytes (j, k) and osteoblasts (l).

Full size image
Figure 7
figure 7

AA-induced GSCs failed to cause tumor in NOD/SCID mice (three mice). Each mouse was injected subcutaneously with 5 × 106 GSCs. Tumor formation in mice was followed up for 3 mo. The results showed that all three mice were free with tumors at injection sites. (a) Mice 1, (b) mice 2, and (c) mice 3.

Full size image

Discussion

Gum tissue plays essential roles in gum disease and mandibular defects. Some studies have hypothesized that GSCs in gum tissue play a role in the overall regeneration of bone augmentation. These cells were considered as a stem cell source for bone regeneration in mandibular defects. In fact, these cells have high potential for osteoblast differentiation. Moreover, they can produce a suitable microenvironment for bone development. These stem cells are also used in craniofacial trauma reconstruction or jawbone before teeth implantation. However, different to umbilical cord blood, umbilical cord gum tissue is a limited tissue source. Therefore, the main obstacle for wide use of GSCs is the limited number of available cells. In this study, we examined stimulation of GSC proliferation by AA.

We isolated GSCs via tissue explant culture and successfully cultured and isolated GSCs with MSC phenotype. These obtained cells satisfied the minimum standard for MSCs according to (Dominici et al. 2006). The isolated cells showed three major characteristics of MSCs, including the following: (1) exhibited fibroblast-like cells that well adhered to the plastic flask surface; (2) expressed specific markers of MSCs including CD44, CD73, CD90, and CD105 positive expression and negative expression for CD14, CD34, CD45, and HLA-DR; and (3) successfully differentiated into adipocytes, osteoblasts, and chondroblasts. These results agreed with previously published studies (Tang et al. 2011; Ge et al. 2012; Hsu et al. 2012; Zhang et al. 2012; Fournier et al. 2013; Gao et al. 2014; El-Sayed et al. 2015; Jin et al. 2015).

We found that GSCs treated with concentrations of AA from 10 to 250 μM showed a concentration-dependent increase of growth proliferation. This conclusion was confirmed by doubling time and cell cycle assay. However, with concentrations higher than AA 250 μM, AA could intoxicate with GSCs that caused them to go to apoptosis. In fact, in this group, almost GSCs go to the G0/G1 phase. In this experiment, we recognized that AA effected to phase S and G2/M. By this chemical, the S and G2/M time clearly reduced.

To the best of our knowledge, this is the first study on the effects of AA on GSC proliferation and stemness. However, the effects of AA on MSCs were confirmed in many previously published studies. AAAA also maintained differentiation potential in bone marrow-derived MSCs through expression of hepatocyte growth factor (Bae et al. 2015). In the adipose-derived stem cell (ADSC) models, AA helps ADSCs against mitoptosis, necroptosis, and apoptosis (Padial-Molina et al. 2015). Vitamin C increased ERK1/2 phosphorylation, and inhibition of the mitogen-activated protein kinase pathway attenuated the proliferation of ADSCs (Kim et al. 2014). By microarray and quantitative polymerase chain reaction analysis, Kim et al. (2014) showed that AA upregulated expression of proliferation-related genes, including Fos, E2F2, Ier2, Mybl1, Cdc45, JunB, FosB, and Cdca5. It also caused increases in the mRNA expression of HGF, IGFBP6, VEGF, bFGF, and KGF (Kim et al. 2014).

Previous studies showed that AA significantly enhanced expression of pluripotent markers such as Sox-2, Oct-4, and Nanog in ADSCs (Yu et al. 2014). In a recent publication, Wei et al. (2014) demonstrated that AA was an essential factor to maintain stemness of mADSCs in vitro (Wei et al. 2014). AA can interact with the C-terminal catalytic domain of Tet enzymes, which likely promotes folding and/or recycling of the cofactor Fe (2+). In mouse embryonic stem cells, AA significantly increases the levels of all 5 mC oxidation products, particularly 5-formylcytosine and 5-carboxylcytosine, leading to a global loss of 5 mC (∼40%) (Yin et al. 2013).

AA also plays an essential role in inducing a pluripotent state in mouse embryonic stem cells by modulating microRNA expression (Gao et al. 2015). In fact, some reports suggest that AA can enhance somatic reprogramming efficiency to produce pluripotent stem cells (Esteban and Pei 2012). The underlying mechanism is related to the increase of promoter activity of pluripotent genes and enhanced protein level (Gao et al. 2014).

However, a few studies have reported AA as a differentiating factor. AA can promote the direct conversion of mouse fibroblasts into beating cardiomyocytes (Talkhabi et al. 2015), induce cardiac differentiation of murine pluripotent stem cells (Ivanyuk et al. 2015), and cause adipocyte differentiation of embryonic stem cells into adipocytes (Cuaranta-Monroy et al. 2014).

Conclusion

GSCs are a new source of stem cells. These stem cells play important roles for teeth as well as jawbone growth. This study showed that AA is a proliferating factor that can trigger GSC proliferation. The proliferation mechanism of AA is related to upregulation of some pluripotent stem cell markers such as SSEA-3, Oct-3/3, Nanog, Sox-2, and TRA-1-60. However, AA-treated GSCs still maintained the MSC phenotype with MSC marker expression as well as mesoderm cell differentiation. More importantly, these cells cannot cause tumors in athymic mice. These results showed that AA can be a suitable factor to stimulate proliferation of GSCs for clinical applications.