Isolation, Culture, and Expansion of MSCs
This study was approved by the Ethics Committee of First Affiliated Hospital, School of Medicine, Zhejiang University. Bone marrow (BM) samples were obtained from the healthy adult donors after taking their consent. The mononuclear cells were collected by density gradient centrifugation (Ficoll 1.077g/mL; Haoyang Biological Manufacture, Co., Ltd., Tianjin, China). The cells were then seeded at a density of 4´105 cells/cm2 in low-glucose Dulbecco’s modified Eagle’s medium (LG-DMEM; Gibco, Carlsbad, CA, USA) addied with 10% fetal bovine serum (FBS; Gibco) and 100 IU/mL penicillin/streptomycin at 37°C in a humidified atmosphere containing 5% CO2. After 48 h, the non-adherent cells were removed and the medium was replaced every 3 days. The cells after reaching 70-80% confluence were trypsinized and reseeded at a density of 8´103 cells/cm2. MSCs at passages 3-4 were used in this study.
R-2HG (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in phosphate buffered saline (PBS). The MSCs were treated with 0 to 1.5mM concentrations of R-2HG.
Cell proliferation analysis
Proliferation of MSCs was determined using Cell Counting Kit-8 assay (CCK-8; Dojin, Tokyo, Japan). Briefly, the cells were plated at the density of 3,000 cells/well in 96-well plates, and then were exposed to R-2HG at a concentration of 0 to 1.5mM. After culturing at 37°C in a humidified incubator with 5% CO2 for 0, 2, 4, 6, or 8 days, the cells were incubated at 37°C with 20 μl CCK-8 solution for 2 h, and the absorbance was measured by a multiwell spectrophotometer (Bio-Rad Laboratories, Tokyo, Japan) at 490 nm.
Flow cytometry assay
MSCs exposed to 0, 1.0, 1.5mM R-2HG were determined by flow cytometry assay. A total of 5×105 cells from single-cell suspensions were incubated for 30 minutes at room temperature with fluorochrome-conjugated monocolonal antibodies against CD34-PE, CD73-APC, CD90-FITC, CD105-PE (eBioscience, San Diego, CA, USA), CD45-FITC, and HLA-DR-PE-Cy5 (Biolegend, San Diego, CA, USA). After washing with PBS, immunofluorescence analysis was performed by flow cytometry using a FACS Calibur system (Beckman Coulter, Miami, FL, USA) and data were calculated using the FlowJo Software. Appropriate isotype-matched antibodies were used as controls.
MSCs were seeded into 0.1% gelatin coated 6-well plates at a density of 10,000 cells/cm2 in LG-DMEM supplemented with 10% FBS. After 2 days, cells were transferred to osteogenic induction medium for 14 days. The medium consists LG-DMEM containing 10% FBS, 10 mM β-glycerophosphate, 0.1 μM dexamethasone and 50 μM ascorbic acid (Sigma-Aldrich). R-2HG at a concentration of 0-1.5 mM was added to the osteogenic induction medium. The medium should be changed for every 3 days. The mineralized areas were revealed using alizarin red staining.
The cells after reaching 80% confluence were trypsinized, washed, and resuspended in high-glucose DMEM with 1 mM sodium pyruvate (Invitrogen), 0.1 μM dexamethasone (Sigma-Aldrich), 200 μM ascorbic acid (Sigma-Aldrich), 1×insulin-transferrin-selenium (Invitrogen) and 10 ng/ml transforming growth factor-1 (Peprotech, London, UK). The viable cells were seeded in 15-ml conical tubes at a density of 5×105 cells per pellet. Next, the cells were gently allowed to centrifuge to the bottom of the tubes to form compact cell pellets, and then incubated in a humidified atmosphere in 5% CO2 at 37°C. The medium should be changed every 3 days. R-2HG at a concentration of 0-1.5mM was used for treatment from day 1.
Sections of paraffin-embedded MSCs pellets were processed for immunohistochemistry using rabbit anti-human collagen type II (Abcam, Cambridge, MA, USA). EnVision detection kit (Dako, Carpinteria, CA) was applied to analyze the immunoreactivity of the sections. Non-immune rabbit- IgG antibody was used as the negative control.
The MSCs were seeded into 6-well plates in a density of 20,000 cells/cm2. While cells were grown to confluence, they were transferred to adipogenic induction medium containing LG-DMED and adipogenic stimulatory supplement (Stem Cell Technologies, Hangzhou, China) and the system was cultured for 21 days. The medium was changed every 3 days. R-2HG at a concentration of 0-1.5 mM was added to the adipogenic induction medium. The adipogenic differentiation was mesured by cellular accumulation of large lipid vacuoles that are stained with oil red O (Sigma-Aldrich).
Real-time Quantitative Polymerase Chain Reaction Analysis
Messenger RNA (mRNA) expressions of osteogenic (BGLAP, IBSP, LPL, SP7), adipogenic (CEBPA, PPARG, ADIPOQ and FABP4) and chondrogenic differentiation related markers (SOX9, RUNX2, COL2A1 and COL10A1) were quantified using real-time quantitative polymerase chain reaction analysis (RT-PCR). The cultured cell layers or pellets of each group were collected on Day 6 of induction medium incubation. The total RNA was extracted from MSCs using Trizol reagent (Invitrogen) and then was reversely transcribed into complementary DNA (cDNA) by PrimeScript RT reagent Kit (Takara, Tokyo, Japan). Equal amounts of cDNA were used and amplified with SYBR Premix Ex Taq using SYBR Premix Ex Taq (Takara). Every sample was performed in in three independent experiments and all the results were normalized to the levels of glyceraldehydes 3-phosphate dehydrogenase (GAPDH). Primer sequences are shown in Table 1.
The expressions of the components of Sonic Hedgehog signaling pathway including Sonic Hedgehog ligand (SHH), Patched 1 (PTCH1), Smoothened (SMO), and Gli transcription factors (GLI-1, 2 and 3) were quantified by RT-PCR. RNA was prepared from MSCs treated in the absence or presence of 1.0 mM and 1.5mM R-2HG during osteogenic induction for 6 days.
Illumina Infinium methylation assay
The changes in DNA methylation of MSCs exposed to R-2HG, and the genome-scale methylation profiles were explored as described previously . MSCs were cultured in proliferation medium in the absence or presence of 1.0 mM R-2HG for 6 days and collected. Bisulfite conversion of genomic DNA was prepared using EZ DNA methylation Kit (Zymo Research, D5002, USA). A total amount of 500 ng of DNA was bisulfite converted and subsequently processed for hybridization onto an Infinium Human Methylation 450 Bead Array (Illumina, San Diego, CA, USA) under the manufacturer’s instructions. This array can interrogate 27,578 CpG dinucleotides encompassing 14,495 genes. In brief, the DNA was mixed with bisulfite, and the nonmethylated C nucleotides were converted to U (T), whereas the methylated C nucleotides remained to be unaffected. Subsequently the bisulfite-treated DNA was amplified, fragmented, and hybridized to locus-specific oligonucleotides on the BeadArray. C or T nucleotides were detected by fluorescence signaling in order to obtain the single-nucleotide extension of the DNA fragments. The results were interpreted as a ratio (β value) of methylated signal (C) when compared with the sum of methylated and unmethylated signal (C-T) for each locus, where 0 was regarded as fully unmethylated DNA and 1 as fully methylated DNA.
To investigate the methylation state of MSCs during osteogenic and chondrogenic differentiation, the cultured cell layers or pellets of MSCs treated either in absence or presence of 1.0 mM R-2HG were collected on Day 6 of induction medium incubation. Bisulfite conversion of genomic DNA was prepared using EZ DNA methylation Kit (Zymo Research, D5002, USA). A total amount of 500 ng of DNA was bisulfite converted and subsequently processed for hybridization onto an Infinium Human Methylation 850 Bead Array (Illumina, San Diego, CA, USA) under the manufacturer’s instructions. Methylation analysis was performed using the R/Bioconductor package Minfi. Methylated CpG sites in promotor region of related genes (BGLAP, IBSP, LPL, SP7, SOX9, RUNX2, COL2A1, COL10A1, SHH, PTCH1, SMO, GLI-1, 2 and 3) were analyzed from the array-based data.
The heat maps were designed by Mev software. The Euclidean distance within the two groups of samples was calculated using the average linkage measure [the mean of all pair-wise distances (linkages) between the members of the two concerned groups]. Gene annotation and enrichment analyses were performed by KEGG databases using the DAVID Bioinformatics Resources (http://david.abcc.ncifcrf.gov/) interfaces and WebGestalt (http://bioinfo.vanderbilt.edu/webgestalt/), respectively.
Gene pathway analysis
To determine the biological processes enriched within genes of differential methylation in the comparisons, we uploaded the gene lists into the Ingenuity Pathway Analysis (IPA; Ingenuity Systems, Redwood City, CA, USA). Each gene symbol was linked to its corresponding gene object in the Ingenuity Pathways Knowledge Base. Then the IPA integrates the genes and molecules that share part of the same biological functions or regulatory networks interacting together. The over-represented cellular and molecular functions were ranked according to the calculated P-value.
The results are expressed as mean ± standard error (SE), each performed in duplicates. Statistical analysis was performed by analysis of variance (ANOVA). All analyses used SPSS software (Paris, France). A p-value of < 0.05 was considered significant.