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The study was approved by the Institutional Review Board of School of Public Health at Jilin University.
Isolation, Culture, and Identification of Human Hair Follicle Stem Cells
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The isolation and culture of hHF-MSCs were performed according to previous studies[4, 16, 17]. The hair follicles were washed with Phosphate-Buffered Saline (PBS) for three times. Then they were transferred into 24-well culture plate which contained one or two follicles per well. The hHF-MSCs were cultured in Dulbecco's Modified Eagle Medium supplemented with F-12 (DMEM/F-12, Gibco by Life technologies, USA, 10% fetal bovine serum (FBS, Gibco, USA) and 2 ng/mL bFGF (Sino Biological Inc, China) at 37 ℃, 5% CO2. The medium was refreshed every 3 days. The cells were digested with 0.25% trypsin (Biosharp, China) when the cell confluence was approximately 80%. Flow cytometry were used to detect the expression of cell surface markers, including CD44 (eBioscience, USA), CD73 (eBioscience, USA), CD90 (eBioscience, USA), CD105 (eBioscience, USA) and CD31 (eBioscience, USA). The assays were performed as described previously[4].
Differentiation Potential of Human Hair Follicle Stem Cells
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For adipogenic differentiation assay, hHF-MSCs were cultured in adipogenic differentiation medium which contained High Glucose Dulbecco's Modified Eagle Medium (DMEM; Gibco, USA) supplemented with 10% FBS (Gibco, USA), 1 μmol/L dexamethasone (Sigma-Aldrich, USA), 0.5 mmol/L isobutyl-methylxanthine (Sigma-Aldrich, USA), 10 μmol/L insulin (Sigma-Aldrich, USA), and 200 μmol/L indomethacin (Sigma-Aldrich, USA). The formation of acellular lipid droplets in hHF-MSCs after two-week culture stained with Oil red O (Sigma-Aldrich, USA) were observed by inversion fluorescence microscope (Leica, German)[5].
For the assay of osteogenic differentiation, hHF-MSCs were cultured in osteogenic differentiation medium which contained High Glucose DMEM (Gibco, USA) supplemented with 10% FBS (Gibco, USA), 0.1 mmol/L dexamethasone (Sigma-Aldrich, USA), 50 mmol/L ascorbate-2-phosphate (Sigma-Aldrich, USA), and 10 nmol/L β-glycerophosphate. The formation of mineralized nodules in hHF-MSCs stained by Alizarinred S (Sigma-Aldrich, USA) after a month culture was observed by inversion fluorescence microscope (Leica, German)[4].
Lentivirus Packaging
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Day 1, 6 × 106 293T cells were cultured in 8 mL High Glucose DMEM (Gibco by Life Technologies, USA) with 10% FBS in a 10 cm dish. Day 2, the lentivirus vector containing NANOG coding region 10 μg, transfection reagents (GeneCopoeia, USA) 22 μL, two packaging plasmids (7.5 μg of psPAX2- gag/pol/tat/rev and 2.5 μg of pMD2.G-VSVG), were added into 1 mL Opti-MEM (Reduced Serum Medium, Gibco by Life Technologies, USA). Six hours later, the medium was replaced for new High Glucose DMEM with 10% FBS for the sake of better growth and lower toxic effect. After 36 h the supernatant was harvested and added into hHF-MSCs supplemented with 10 μg/mL polybrene (Santa, USA) and 2 ng/mL bFGF (Sino Biological Inc, China). Seventy-two hours later, the transduction efficiency was observed by inversion fluorescence microscope (Leica, German) and also was verified by Western blot.
Assessment of Cell Proliferation
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hHF-MSCs were seeded at 5 × 103 cells per well in a 96-well plate with culture medium containing 100 μL DMEM/F-12 and 10% FBS without bFGF and cultured for 5 days. Each day, 10 μL of 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide (MTT, 5 mg/mL, Dingguo, China) were added into culture medium and incubated at 37 ℃, 5% CO2 in dark for 4 h. After removing the medium, 150 μL dimethyl sulfoxide were added into each well. Five minutes later, the MTT absorbance could be measured by microplate reader (SYNERGY H1 microplate reader, BioTek, USA) at 490 nm.
Assessment of Cell Survival Rate
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CCK-8 was used to assess the cell survival rate of hHF-MSCs treated with H2O2. hHF-MSC cells were plated in a 96-well plate and when the confluence of the cells was 80%, the cells were cultured with the medium supplemented with 0.5% FBS for 24 h. 24 h later, the concentration of FBS in the medium was changed to 1% and the cells were treated with different concentrations of H2O2 (0, 100, 200, 400, 800 μmol/L) at 37 ℃ with 5% CO2 for 2 h. Two hours later, the culture media was removed and 100 μL fresh culture medium and 10 μL CCK8 (Fuyuanbio, China) were added to the cells and cultured at 37 ℃ for 2 h. The absorbance was determined by microplate reader (SYNERGY H1 microplate reader, BioTek, USA) at 450 nm. Cell survival rate = (OD H2O2 damage-OD blank) / (OD control-OD blank) × 100%. In addition, another experiment was performed to explore the damage of 400 μmol/L H2O2 to the cells at different time (0, 1, 2, 4, 6 h) according to the CCK-8 assay above.
Assay of Intracellular ROS Production
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CM-H2DCFDA (General Oxidative Stress Indicator, Invitrogen, USA) was used to measure intracellular ROS production through fluorescence intensity. hHF-MSC cells were plated in a 96-well plate and incubated with 10 μmol/L CM-H2DCFDA at 37 ℃ for 30 min in darkness. Then cells were treated with 400 μmol/L H2O2 for 2 h after being washed with DMEM/F-12 medium without FBS three times. Next, the fluorescence intensity was detected by Cytation 3 Cell Imaging Multi-Mode Reader (BioTek, USA). The excitation wavelength and emission wavelength were 488 nm and 525 nm respectively.
Cell Apoptosis Determination by Flow Cytometry
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Annexin V-FITC Apoptosis Analysis Kit (Sungene, Tianjin, China) was used to detect the cell apoptosis. Cells were collected and cell concentration was adjusted to 1 × 106/ tube, rinsed by precool 4 ℃ PBS and resuspended by 100 μL binding buffer, 5 μL Annexin-V FITC was added to the tube and tube was shaken gently, then incubated at room temperature in darkness for 10 min, 10 min later, 5 μL PI solution was added to the cells and incubated for 5 min, finally 400 μL PBS was added to the tube and the cells were detected (BD, FACSCalibur, USA).
Western Blot
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To measure relative protein level, western blots were performed according to previous study of our team[4]. The antibody information was AKT (pan) (C67E7) Rabbit mAb (CST, USA, 1:1, 000), phospho-AKT (Ser473) Rabbit mAb (CST, USA, 1:2, 000), p44/42 MAPK (Erk1/2) Rabbit mAb (CST, USA, 1:1, 000), Phospho-p44/42 MAPK (Erk1/2) Rabbit mAb (CST, USA, 1:1, 000), NANOG Rabbit mAb (CST, USA, 1:1, 000), p21Waf1/Cip1 (12D1) Rabbit mAb (CST, USA, 1:1, 000), GAPDH Mouse mAb (ProteinTech, USA, 1:10, 000), HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (ProteinTech Group Inc, USA, 1:5, 000), and HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (ProteinTech Group Inc, USA, 1:5, 000). The images were obtained by Chemiluminescence imaging analysis system (ECL from Tanon 5, 200, Shanghai, China) and band intensity was analyzed by Tanon Gis analytical software (Shanghai, China).
The Inhibitor LY294002 to Block PI3K/AKT Pathway
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For western blot, NANOG hHF-MSCs were seeded into a 10 cm plate while for cell survial rate and ROS, the cells were seed in a 96-well plate. When the confluence of the cells was 80%, inhibitor LY294002 was added to the cells at the final concentration of 40 μmol/L for 24 h. The inhibitor LY294002 (MedChem Express, USA) was used to inhibiting PI3K/AKT pathway or blocking AKT phosphorylation. The optimal treatment concentration and time for LY294002 were determined by western blotting (data not shown).
Statistical Analysis
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Software SPSS version 24.0 (SPSS, Chicago, IL) was used for statistical analysis and all quantitative data was presented as mean ± SD. All data are from at least three independent experiments. Comparisons between two groups were tested by Student's t-test. P < 0.05 was considered statistically significant.