Cell isolation and culture
SHED were isolated and collected from non-caries exfoliated human deciduous teeth of 4 to 10 years old children after inform consent after inform consent, and approved by the Ethics Committee of Hospital of Stomatology in Sun Yat-sen University. Briefly, the pulps from deciduous teeth were minced and digested with 3 mg/ml collagenase type I and 4 mg/ml dispase (Gibco-BRL, USA), and cultured using DMEM (Gibco-BRL, USA) contained 10% FBS, 100 units/ml streptomycin (HyClone, USA), 100 units/ml penicillin (HyClone, USA). hBMSCs were bought from Cyagen Biosciences (China) and cultured using DMEM contained 10% FBS, 100 units/ml streptomycin and 100 units/ml penicillin. Human umbilical vein endothelial cells (HUVECs) were bought from the China Center for Type Culture Collection (CCTCC, China) and cultured using endothelial cell medium (ECM, ScienCell, USA) contained 5% FBS, 100 units/ml streptomycin and 100 units/ml penicillin. Hepatocyte growth factor (HGF), stem cell factor (SCF), insulin-like growth factor 2 (IGF2) (all from PeproTech, USA) were supplemented into DMEM during the long-term expansion of hBMSCs from passage 3 (P3) to passage 8 (P8).
Supernatant collection
SHED or hBMSCs (P3) were seeded into a 10-cm dish (4 × 105 cells per dish) and cultured in DMEM contained 10% FBS for 3 days. The supernatants of SHED or hBMSCs were collected (centrifuged at 800 g for 5 min to remove cell debris) and preserved at -80 °C. For supplying enough nutrition to cells, the supernatants were mixed with fresh DMEM contained 10% FBS in a 1:1 ratio when used, named as conditioned medium from SHED (SHED-CM) or conditioned medium from hBMSCs (hBMSCs-CM). To explore the effect of conditioned mediums on stemness and senescence of hBMSCs during long-term expansion, hBMSCs were cultured in DMEM, SHED-CM or hBMSCs-CM from P3 to P8 respectively.
Colony-forming unit (CFU) assay
To detect the effect of conditioned mediums on the self-renewal ability of hBMSCs (P3), 1000 cells were seeded into a 10-cm dish in each group and cultured in DMEM, SHED-CM or hBMSCs-CM for 14 days. hBMSCs were washed with PBS, fixed with 4% paraformaldehyde (PFA) and stained with 0.1% (w/v) crystal violet (Sigma, USA).
Cell cycle analysis
hBMSCs in each group (P3, P8, P8-SHED-CM and P8-hBMSCs-CM) were seeded into 6-well culture plates (5 × 104 cells per well) and collected after 3 days. Cells were washed with cold PBS and fixed in 70% ethanol overnight at 4 °C. After being washed with cold PBS, cells were incubated with 50 μg/ml propidium iodide (PI), 100 μg/ml ribonuclease A and 0.1% TritonX-100 in PBS at 4 °C for 30min, and detected by flow cytometry (Beckman Coulter, Germany). Data were analyzed by Flowjo software.
β-galactosidase staining
hBMSCs in each group (P3, P8, P8-SHED-CM and P8-hBMSCs-CM) were seeded into 6-well culture plates (5 × 104 cells per well) and cultured for 3 days. Senescent cells were stained by a β-galactosidase (β-gal) staining kit (Beyotime, China) according to the manufacturer’s instructions. The β-gal-positive cells in each group were scanned by microscopy and calculated.
Osteogenic induction
For RT-qPCR and Western blot assay, hBMSCs in each group were cultured into 6-well plates (5 × 104 cells per well) for 3 days, followed by induced with osteogenic differentiation medium (DMEM contained 10% FBS, 10 mM β-glycerophosphate, 10 nM dexamethasone and 50 μg/mL ascorbic acid) for 7 days. For alizarin red and alkaline phosphatase (ALP) staining, hBMSCs in each group were cultured into 24-well plates (1 × 104 cells per well) for 3 days, followed by induced with osteogenic differentiation medium for 14 days. P3 hBMSCs without osteogenic induction were named as “P3”, with induction named as “P3+”. Similarly, other groups were named by the same way (as P8, P8+, P8-SHED-CM+, P8-hBMSCs-CM+).
Total RNA extraction and reverse transcription quantitative PCR (RT-qPCR)
Total RNA of cells was isolated with RNA isolation kit (Ultrapure RNA Kit, CW Biotech, China) and transcribed into cDNA using Reverse Transcriptase M-MLV kit (TaKaRa, China). RT-qPCR assay was performed according to the manufacturer’s instructions of SYBR Green PCR Master Mix kit (Roche, Switzerland). GAPDH was used as the internal control. The cycling conditions were all as follows: incubation at 95°C for 10 min, 40 cycles of denaturation at 95°C for 15s, annealing at 60°C for 20s and extension at 72°C for 20s. The primer sequences for each gene are listed in Table 1. The expression level of targeted gene was analyzed by 2−ΔΔCt method.
Table 1 Primer sequences used in reverse transcription quantitative PCR (RT-qPCR).
Gene
|
Sequence
|
p16
|
Forward: 5’-CCCCTTGCCTGGAAAGATAC-3’
Reverse: 5’-AGCCCCTCCTCTTTCTTCCT-3’
|
p21
|
Forward: 5’-AGCAGCGGAACAAGGAGT-3’
Reverse: 5’-TTACAGTCTAGGTGGAGAAACG-3’
|
Nanog
|
Forward: 5’-AAGGCCTCAGCACCTACCTA-3’
Reverse: 5’-TGCACCAGGTCTGAGTGTTC-3’
|
OCT4
|
Forward: 5’-TGGATGTCAGGGCTCTTTGTC-3’
Reverse: 5’-ACCTTCCCAAATAGAACCCCC-3’
|
Runx2
|
Forward: 5’-TGGTTACTGTCATGGCGGGTA-3’
Reverse: 5’-TCTCAGATCGTTGAACCTTGCTA-3’
|
BSP
|
Forward: 5’-GAACCACTTCCCCACCTTTTG-3’
Reverse: 5’-ATTCTGACCATCATAGCCATCG-3’
|
ALP
|
Forward: 5’-TTCAAACCGAGATACAAGCACT-3’
Reverse: 5’-GGGCCAGACCAAAGATAGAG-3’
|
GAPDH
|
Forward: 5’-GGAGCGAGATCCCTCCAAAAT-3’
Reverse: 5’-GGCTGTTGTCATACTTCTCATGG-3’
|
Western blot analysis
Cells were harvested and lysed in RIPA (50mM Tris-HCl pH7.4, 150mM NaCl, 1%TritonX-100, 0.5% sodium deoxycholate, 0.1% SDS and protease inhibitor cocktail) on ice for 30 min. Total protein concentrations were measured by a BCA protein assay kit (Pierce, Thermo Scientific). Western blot assay was performed using protocols described below. Briefly, 40μg total protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto nitrocellulose (NC) membranes, blocked by 5% (w/v) nonfat milk for 1 h at room temperature and then incubated with primary antibodies specific for Runx2 (ABclonal, 1:500), BSP (Boster, 1:200), Mitofusin 1 (Mfn1, Boster, 1:200), Mitofusin 2 (Mfn2, Boster, 1:200), Catalase (Cell Signaling Technology, 1:1000), SOD2 (Cell Signaling Technology, 1:1000), PI3K (Boster, 1:200), phospho-AKT (p-AKT, Cell Signaling Technology, 1:1000), AKT (Cell Signaling Technology, 1:1000), phospho-ERK1/2 (p-ERK1/2, Cell Signaling Technology, 1:2000), Erk1/2 (Cell Signaling Technology, 1:1000), phospho-STAT3 (p-STAT3, Cell Signaling Technology, 1:2000), STAT3 (Cell Signaling Technology, 1:2000) and β-actin (Sigma 1:5000) overnight at 4°C. After incubated with secondary antibodies for 1 h at room temperature, the membranes were scanned by an Odyssey two-color infrared laser imaging system (LI-COR Biosciences, USA). Relative protein expression levels were analyzed by ImageJ software and normalized to β-actin.
Alizarin red and ALP staining
For alizarin red staining (ARS), hBMSCs in each group (P3, P3+, P8, P8+, P8-SHED-CM+, P8-hBMSCs-CM+) were fixed with 4% PFA and stained with 1% (w/v) Alizarin Red. The nodules were scanned by microscopy and dissolved by 10% (w/v) cetylpyridinium chloride (Sigma, USA). OD values were measured at 562 nm. ALP staining was performed by the BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime Biotech, China), according to the manufacturer’s instructions.
Tube formation assay in vitro
The supernatants of hBMSCs in each group (P3, P8, P8-SHED-CM and P8-hBMSCs-CM) were collected after 3 days cultured in DMEM. Tube formation assay were performed as below to evaluate the pro-angiogenic capacity of hBMSCs. Briefly, a 48-well culture plate was prepared with 100 μl matrigel matrix mixture (mixed with serum-free DMEM at a ratio of 1:1) (BD Biosciences, USA) per well in advance. Then, HUVECs were resuspended by the supernatants of hBMSCs, and seeded into plates at 6 × 104 cells per well. After 4 h incubation at 37 °C, 5% CO2, tube formation in each group was detected under microscopy. The results of tube length, tube area, and the number of branch points were analyzed by ImageJ software.
Cell proliferation detection
The effect of HGF, SCF and IGF2 on the proliferation ability of hBMSCs was detected using CFSE assay. hBMSCs were labeled with 1 μM of CFSE (Invitrogen, USA) before culture, then hBMSCs were seeded into plates and cultured in DMEM supplemented with different factors at different concentrations for 3 days. The proliferative cell rate was detected by flow cytometry (CytoFLEX, Beckman Coulter). Data were analyzed by Flowjo software.
Assessment of ROS production
Intracellular ROS was detected by fluorescence probe DCFH-DA (Sigma,USA) according to the manufacturer’s protocol. Briefly, cells were incubated with 10 μM DCFH-DA in dark at 37 °C for 20 minutes, then washed by PBS and observed under fluorescence microscope (Cael Zeiss, Oberkochen, Germany). To obtain quantitative result, the fluorescence intensity was detected by flow cytometry (CytoFLEX, Beckman Coulter). Data were analyzed with CytExpert Software (Beckman Coulter).
Mitochondrial membrane potential assay
Mitochondrial membrane potential was detected by a JC-1 fluorescent probe assay kit (Beyotime Biotech, China) according to the manufacturer’s protocol. Cell fluorescence was observed under fluorescence microscope (Cael Zeiss, Oberkochen, Germany). To obtain quantitative results, the fluorescence intensity was detected by flow cytometry (CytoFLEX, Beckman Coulter). Data were analyzed with CytExpert Software (Beckman Coulter).
Statistical analysis
All data were performed in triplicate and results were expressed as mean ± SD. Statistical analysis was conducted using SPSS20.0 software package (SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) was used and post-hoc Bonferroni test was performed for multiple comparisons. P values < 0.05 were considered statistically significant.