Tooth sample collection
Human impacted third molars (n=7) were collected from Thai patients (15-20 years) at the Faculty of Dentistry, Mahidol University, Thailand. The ethical consideration and research protocol were approved by the Ethics Committee on Human Rights Related to Human Experimentation of Faculty of Dentistry/Faculty of Pharmacy, Mahidol University (COE. No. MU-DT/PY-IRB 2019/027.2405). The inclusion criteria of the teeth consists of the presence of apical papilla tissue, caries-free, and no sign of pulp necrosis, trauma, or periodontal disease.
Cell isolation and culture
The isolation of hSCAPs was performed by the enzymatic-disaggregation method as previously described (6). Briefly, the teeth were collected in a proliferating medium consisting of Alpha Minimum Essential Medium (αMEM, Gibco, Life Technologies, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (FBS, Gibco, Life Technologies), 100 U/mL Penicillin, and 100 μM/mL Streptomycin (Gibco, Life Technologies), and washed with 0.1 M phosphate buffer saline (PBS, Sigma-Aldrich, St. Louis, MO, USA). Following teeth extraction, the apical papilla tissue was separated, dissected into smaller pieces, and digested with a cocktail of 3 mg/mL collagenase type I (Worthington, Lakewood, NJ, USA), and 4 mg/mL dispase II (Sigma-Aldrich) at 37 oC for 1 hour. The digested tissue was filtered through 70 µm cell strainer (FalconTM, Fisher Scientific, Waltham, MA, USA), seeded into a cell culture vessel (T-75 cm2 flask, NuncTM, Thermo Scientific, Waltham, MA, USA), and cultured in the proliferating medium at 37 oC, 5% CO2, and 95% humidity incubator. The medium was changed every 2 days until confluence was achieved. Then, the cells were subculture to expand the cell population. Cells at passages 2-6 were used in this study.
Cell surface molecule marker analysis
The uncharacterized cells (1 x 106 cells) were harvested and the cell surface antigen molecules were analyzed by BD FACS Canto Flow cytometer (BD Biosciences, San Jose, CA, USA). The cells were detected for MSCs specific markers using antibodies as follows: anti-human CD73 (APC/Cy7) (Biolegend, San Diego, CA, USA), anti-human CD90 (PE) (Biolegend), anti-human CD105 (Alexa Flour® 488) (Biolegend), and anti-human CD146 (PerCP/Cy5.5) (Biolegend). An antibody against hematopoietic stem cell marker, anti-human CD34 (APC) (Biolegend), was used as a negative control. The level of cell surface antigen molecules expression was analyzed using the BD FACSDivaTM software (BD Biosciences).
Colony-forming unit fibroblast
The uncharacterized cells were seeded in triplicate into 6-well plates (NuncTM, Thermo Scientific) at a density of 500 cells/well, and cultured in the proliferating medium for 12 days. The medium was changed every 2 days. The colonies of these cells were visualized by Giemsa staining, and captured by the Compact Cell Culture Microscope, CKX3 (Olympus, Hamburg, Germany).
The uncharacterized cells were seeded in 24-well plates (NuncTM, Thermo Scientific) at a density of 2 x 104 cells/well, and cultured in the proliferating medium until reaching 80% confluence. Osteogenic differentiation was induced by culturing for 4 weeks in an osteogenic induction medium consisting of 0.1 µM dexamethasone (Sigma-Aldrich), 50 µg/mL ascorbate-2-phosphate (Sigma-Aldrich), 10 mM β-glycerophosphate (Sigma-Aldrich) in αMEM, 10% FBS, 100 U/mL Penicillin, and 100 μM/mL Streptomycin. The medium was changed every 2 days. The calcification of an extracellular matrix was observed with Alizarin red staining, and captured by the Compact Cell Culture Microscope, CKX3 (Olympus).
The uncharacterized cells were seeded in 24-well plates (NuncTM, Thermo Scientific) at a density of 2 x 104 cells/well, and cultured in the proliferating medium until reaching 100% confluence. Adipogenic differentiation was induced by culturing for 6 weeks in an adipogenic induction medium consisting of the proliferating medium supplemented with 1 µM dexamethasone (Sigma-Aldrich), 50 µM Indomethacin (Sigma-Aldrich), 1 µg/mL Insulin (Sigma-Aldrich), and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX, Sigma-Aldrich). Oil Red O was stained to visualize lipid droplets, and captured by the Compact Cell Culture Microscope, CKX3 (Olympus).
Cell viability of resveratrol treated hSCAPs
The cell viability of resveratrol treated hSCAPs was performed by the methylthiazolyldiphenyl-tetrazolium bromide (MTT, Sigma-Aldrich) assay. Resveratrol (trans-3, 4’, 5-trihydroxystibene; Sigma-Aldrich) was freshly prepared as a 100 µM stock solution by diluting with αMEM, 100 U/mL Penicillin, 100 μM/mL Streptomycin, and maintained in dark condition. The characterized hSCAPs were seeded in 96-well plates (NuncTM, Thermo Scientific) at a density of 1 x 104 cells/well. After 24 hours, the hSCAPs were treated with different concentrations of resveratrol (0, 5, 10, 15, 25, 50, and 100 µM) for 6, 12, and 24 hours. Then, the MTT assay was performed. The MTT working solution (0.5 mg/mL) was added, and the plates were incubated for an additional 2 hours at 37 oC. After centrifugation, the solution was replaced with dimethyl sulfoxide (DMSO, Fisher Scientific). The absorbance of each well at 570 nm and 690 nm was measured with a micro-plate reader (Epoch, Fisher Scientific, Waltham, MA, USA). The percentage of cell viability of hSCAPs in resveratrol treatments (A570-A690 of experimental group x 100/ A570-A690 of control group), (n=5) and the 50% inhibitory concentration (IC50) of resveratrol pre-treatment on hSCAPs were reported.
Optimal condition of resveratrol pre-treatment
The hSCAPs were seeded in 6-well plates at a density 1 x 105 cells/well. After 24 hours, the cells were incubated with different non-cellular toxicity concentrations of resveratrol for 12 hours, and qRT-PCR was performed to select the concentration of resveratrol that induced the highest NES expression of hSCAPs. Then, the concentration was used to assess NES expression at various incubation times (1, 6, 12, and 24 hours). The treatments were also investigated for morphological change with β-III tubulin immunocytochemistry staining. The hSCAPs treated with resveratrol at the concentration and incubation time that brought the highest NES expression will be termed “RSV-hSCAPs”. The RSV-hSCAPs were validated the neuronal genes profiling with SOX1, PAX6, and immunofluorescences with Ki67, neurofilaments (NF), and were further induced into neuronal differentiation.
The hSCAPs were seeded on poly-d-lysine (Sigma-Aldrich) coated cover slips (Electron Microscopy Sciences, Hatfield, PA, USA) in 6-well plates at a density 1 x 105 cells/well, and pre-incubated with the optimal condition of resveratrol (RSV-hSCAPs) or without resveratrol (hSCAPs). Then, both hSCAPs and RSV-hSCAPs were exposed to 2 phases of neuronal induction medium. First, the cells were incubated with Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (Ham) (DMEM/F-12, Gibco, Life Technologies) supplemented with 10% FBS, 100 U/mL Penicillin, 100 μM/mL Streptomycin, 10 ng/mL basic fibroblast growth factor (bFGF, Gibco Life Technologies), and 500 µM β-mercaptoethanol (Sigma-Aldrich) for 24 hours. After that, the cells were induced into a phase II neuronal induction medium which consisted of DMEM/F-12, 100 U/mL Penicillin, 100 μM/mL Streptomycin, 2% DMSO, and 100 µM butylated hydroxyanisole (BHA, Sigma-Aldrich) for 6 hours. The negative control hSCAPs (crt-hSCAPs) was pre-incubated for 12 hours with αMEM, 100 U/mL Penicillin, 100 μM/mL Streptomycin, and then cultured with DMEM/F-12, 10% FBS, 100 U/mL Penicillin, and 100 μM/mL Streptomycin for 24 hours. The medium was then replaced with DMEM/F-12, 100 U/mL Penicillin, and 100 μM/mL Streptomycin for 6 hours.
The specimens were fixed in 4% paraformaldehyde (Sigma-Aldrich) in PBS at room temperature for 1 hour, followed by 20% ice-cold methanol (Sigma-Aldrich) in PBS for 5 minutes, then washed with PBS. Subsequently, the specimens were permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) in PBS overnight at 4 oC, and blocked with 15% bovine serum albumin (BSA, Sigma-Aldrich) at 4 oC for 12 hours. The specimens were incubated overnight at 4 oC with anti-mouse Nestin antibody (Biolegend) at a dilution of 1: 500, anti-mouse β-III tubulin antibody (Biolegend) at a dilution of 1: 1,000, anti-mouse Ki67 (Developmental Studies Hybridoma bank, Iowa City, IA, USA) at a dilution 1: 100, anti-mouse NF (Developmental Studies Hybridoma bank) at a dilution 1: 100 which diluted with 5% BSA in PBS with 0.05% Tween-20 (Sigma-Aldrich). Then, the specimens were conjugated with goat anti-mouse IgG highly cross-adsorbed secondary antibody, Alexa Fluor plus 488 (Invitrogen, New York, NY, USA) at a dilution of 1: 1,000 at room temperature for 4 hours. Nuclei were counterstained, and mounted with ProLongTM Diamond Antifade Mountant with DAPI (Invitrogen). The samples were visualized, and captured by the Digital Fluorescence Microscope, BX53 (Olympus). The percentage of neuronal differentiation (the number of differentiated cells x 100/total cells) was quantified using the ImageJ program (NIH, Bethesda, MD, USA) by random counting (n=5).
Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)
Total RNA was extracted using the Nucleospin RNA plus kit (Macherey-Nagel, Bethlehem, PA, USA), and converted into cDNA using iScript RT Supermix (Bio-Rad, Hercules, CA, USA). The qRT-PCR was performed using KAPA SYBR® FAST qPCR kits (Sigma-Aldrich) with CFX96TM real-time PCR detection system (Bio-Rad). The qRT-PCR reaction conditions were 95 oC for 3 minutes, followed by 40 cycles of 95 oC for 3 seconds, 52 oC for 30 seconds. The interesting primers (Integrated DNA Technologies, Gemini Singapore Science Park II, Singapore) used in this study are listed in Table 1. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control, and the expression of interested genes was measured by 2^-ΔΔCt method (22).
Cresyl violet staining
The specimens were fixed in 4% paraformaldehyde at room temperature for 60 minutes, then washed with PBS for 5 minutes and double distill water (ddH2O) for 1 minute. Subsequently, the specimens were incubated with Cresyl Violet Acetate working solution (Electron Microscopy Sciences) at dark condition for 60 minutes. Then, the specimens were washed with ddH2O, followed by serial dehydration of 90%, 95%, and 100% ethanol respectively. Cell imaging were captured by the Compact Cell Culture Microscope, CKX3 (Olympus).
Intracellular calcium oscillation
In order to identify potential of neuronal differentiation, we evaluated calcium influx which is an indicator for neurotransmitter transmission. The intracellular calcium assessment was described in previous study (23). The specimens were incubated with 3 µM Fluo-3 AM (Invitrogen) and 0.08% pluronic acid (Invitrogen) in DMEM/F-12, 100 U/mL Penicillin, 100 μM/mL Streptomycin at 37 oC for 60 minutes. Subsequently, the specimens were washed with DMEM/F-12, 100 U/mL Penicillin, 100 μM/mL Streptomycin, and PBS. The specimens were maintained in Tyrode’s solution (5 mM KCl, 129 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 30 mM glucose, and 25 mM HEPES, pH 7.4) (All from Sigma-Aldrich). The neurotransmitter releasing ability of differentiated cells was simulated with 50 mM KCI. The intensity of calcium was recorded time-lapse at excitation 506 nm for 3 minutes by the Live-cell fluorescence microscope, IX83XDC (Olympus), and interpreted using the ImageJ program (NIH).
The individual experiment was repeated 3 times. The data were expressed as the mean ± standard error of mean (SEM), the difference between the experimental groups and the control group were compared using Tukey's Multiple Comparison Test via GraphPad Prism version 5.00.288 (San Diego, CA, USA). The differences with *p-value < 0.05, and ***p-value < 0.001 were considered significant.