Culture and differentiation of hMSCs
hMSCs harvested from normal human bone marrow were purchased from Lonza (Walkersville, MD, USA) at passage 2. Cells were expanded for no more than five passages in “mesenchymal stem cell growth medium” (MSCGM; Lonza) at 37 °C in a humidified atmosphere containing 7.5 % CO2. Studies were performed with hMSCs from three different donors, encoded 5F0138, 6F4085, and 7F3458.
For differentiation experiments, 4.0 × 104 cells per cm2 were seeded in high-glucose-containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (a selected lot from Lonza), 100 U/ml penicillin, and 100 μg/ml streptomycin. This medium will be further referred to as “proliferation medium” (PM). The next day, cells were switched to either osteogenic or adipogenic differentiation medium. Cells treated with PM were used as negative controls. Osteogenic differentiation medium (ODM) was composed of PM supplemented with 10−7 M DEX, 0.2 mM ascorbate, and 10 mM β-glycerophosphate. Adipogenic differentiation medium (ADM) was composed of PM supplemented with 10−6 M DEX, 10 μg/ml insulin (R&D Systems, Minneapolis, MN, USA), 10−7 M rosiglitazone (Sigma-Aldrich, St. Louis, MO, USA), and 500 μM IBMX (Sigma-Aldrich). Unless indicated otherwise, recombinant human TGFβ1 and BMP2 (both from R&D Systems) were used at concentrations of 2 ng/ml and 125 ng/ml, respectively. Prostaglandin E2 (PGE2), Batimastat, and Zopolrestat (Sigma-Aldrich) were used at the indicated concentrations. Media were refreshed every 3–4 days.
Alkaline phosphatase assays
To quantify alkaline phosphatase (ALP) enzymatic activity as a measure of osteogenic differentiation, hMSCs were seeded in 96-well tissue culture plates as already described, after which cells were differentiated for 7 days in osteogenic differentiation medium. ALP enzymatic activity was quantified by measuring the formation of p-nitrophenol from p-nitrophenyl phosphate (PNPP; Sigma-Aldrich), as described previously [13]. ALP enzymatic activity was corrected for differences in cell number, as determined by a Neutral Red assay. Cells were incubated with Neutral Red dye diluted in PBS for 1 hour at 37 °C. After washing with PBS, the dye was extracted from the cells using 0.05 M NaH2PO4 in 50 % EtOH, after which the absorbance was measured at 540 nm.
For histochemical analysis of ALP activity, cells were seeded in 48-well tissue culture plates and differentiated for 7 days in osteogenic differentiation medium. Subsequently, cells were fixed in 3.7 % formaldehyde/PBS for 10 min at 22 °C. After washing with PBS, cells were incubated for 1 hour at 37 °C in a mixture of 0.1 mg/ml naphtol AS-MX phosphate (Sigma-Aldrich), 0.5 % N,N-dimethylformamide, 2 mM MgCl2, and 0.6 mg/ml Fast Blue BB salt (Sigma-Aldrich) in 0.1 M Tris–HCl, pH 8.5.
Mineralization assay
To measure calcium deposition in the extracellular matrix, hMSCs were seeded in 24-well tissue culture plates and cultured for 13 days under osteogenic or adipogenic differentiation conditions, as indicated. Cells were subsequently washed twice with PBS after which calcium was extracted from the extracellular matrix by treatment with 150 μl of 0.5 M HCl. Calcium concentrations were measured in a colorimetric assay using o-cresolphtalein complexone as a chromogenic agent, according to the protocol provided by the manufacturer (Sigma-Aldrich).
Oil Red O staining and Triglyceride assay
To quantify adipogenic differentiation, lipid droplets were stained in mature adipocytes obtained after treatment of hMSCs for 9 days in adipogenic differentiation medium. Cells were first washed twice with PBS, fixed for 30 min with 1 % formaldehyde in PBS, and then washed once with water and twice with 60 % isopropanol. Cells were then stained for 1 hour with 0.3 % w/v Oil Red O (Sigma-Aldrich) in 60 % isopropanol. Subsequently, cells were washed once with 60 % isopropanol and twice with distilled water. For quantification of Oil Red O staining, samples were treated with 100 % isopropanol and absorbance was measured at 530 nm.
The amount of triglycerides stored in lipid droplets of mature adipocytes was quantified after treatment of hMSCs for 9 days in adipogenic differentiation medium. Cells in 96-well tissue culture plates were washed twice with PBS. Triglycerides were extracted from the lipid droplets by freezing the cells in 50 μl of a buffer containing 25 mM Tris–HCl (pH 7.5) and 1 mM EDTA, followed by addition of 40 μl tert-butanol and 10 μl methanol. Samples were heat-dried at 55 °C, after which they were resuspended in Triglycerides LiquiColor® mono reagents (HUMAN GmbH, Wiesbaden, Germany). Triglycerides were quantified by measuring the absorbance at 490 nm.
RNA isolation and real-time quantitative RT-PCR
RNA was isolated as described by Piek et al. [13]. For cDNA synthesis, 1 μg of total RNA was reverse transcribed using random hexamer primers and SUPERSCRIPT™ II reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Subsequently, cDNA was amplified in a quantitative real-time PCR, performed using Power SYBR Green® PCR Mastermix (Applied Biosystems, Foster City, CA, USA) on an Applied Biosystems 7500 Real-time Fast PCR System. For each gene, PCR was carried out in duplicate and mean expression values were calculated relative to the mean expression level of the housekeeping gene RPS27A (ribosomal protein S27a). Human gene-specific PCR primers used included the following:
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Histone deacetylase 5 (HDAC5)-FW: 5′-ATGACAACGGGAACTTCTTTCC-3′.
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Histone deacetylase 5 (HDAC5)-RV: 5′-CCATGCCACGTTCACATTGTA-3′.
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Adiponectin (ADIPOQ)-FW: 5′-CCCAAAGAGGAGAGAGGAAGC-3′.
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Adiponectin (ADIPOQ)-RV: 5′-GCCAGAGCAATGAGATGCAA-3′.
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Alkaline phosphatase (ALPL)-FW: 5′-GATGGACAAGTTCCCCTTCGT-3′.
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Alkaline phosphatase (ALPL)-RV: 5′-GGACCTGGGCATTGGTGTT-3′.
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Ribosomal protein S27a (RPS27A)-FW: 5′-GTTAAGCTGGCTGTCCTGAAA-3′.
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Ribosomal protein S27a (RPS27A)-RV: 5′-CATCAGAAGGGCACTCTCG-3′.
Immunoblotting
hMSCs were seeded at 4.0 × 104 cells per cm2 in six-well plates and cultured for 24 hours in the indicated differentiation media. Cells were then lysed in 250 μl of RIPA lysis buffer per well. Then 5 μl of reducing sample buffer was added to 25 μl of lysate, heated to 95 °C, and subsequently loaded onto an 8 % SDS-PAGE gel. HDAC5 was detected on blots using a goat polyclonal anti-HDAC5 antibody (G-18, 1/200 dilution) raised against the N-terminus of human HDAC5 (sc-5250; Santa Cruz Biotechnology, Dallas, TX, USA), followed by an HRP-labeled polyclonal secondary antibody. Antibodies against α-tubulin (Sigma) served as a loading control.
Gene expression microarray analysis
To identify genes that are regulated during osteogenic and adipogenic differentiation of hMSCs, a total of 54 samples (each containing 800,000 cells/ 20 cm2) were seeded in PM and grown for 24 hours. Subsequently the medium was exchanged for differentiation medium, now consisting of PM with 10−6 M DEX, 10 μg/ml insulin, 10−7 M rosiglitazone, and 50 ng/ml BMP2 (B). In addition, either 5 ng/ml TGFβ (BT), or 250 μM IBMX (BI), or 5 ng/ml TGFβ and 250 μM IBMX (BTI) were added. Samples were incubated for either 0, 1, 2, 3, or 7 days. Experiments for each group and time point were carried out as three biological replicates, while the untreated control group (time 0) consisted of six samples. RNA was isolated as already described, and hybridized onto Affymetrix HGU 133 plus 2.0 microarrays according to existing protocols [13].
Microarray data were analyzed with the R language for statistical computing using appropriate Bioconductor packages (http://bioconductor.org/) for reading, normalizing, and statistically evaluating the data, followed by annotation of the gene sets and integration of parallel data sources. Briefly, the analysis started with a careful quality assessment of the dataset using the automatic R pipeline AffymetrixQC [14], which was customized and run locally. All 54 microarrays passed the quality control and were included in the analysis, consisting of robust microarray analysis (RMA) normalization [15], followed by statistical analysis to find differentially expressed genes using Linear Models for Microarray Data (LIMMA) [16], and subsequent functional annotation and enrichment analysis using the online resource Database for Annotation, Visualization and Integrated Discovery (DAVID) [17, 18]. Finally, the list of differentially expressed genes for the contrasts of interest was crossed with the information from the DrugBank database [19] in order to derive the final list of candidate genes for experimental testing. All R scripts used for this analysis are available upon request. Current microarray data have been deposited in NCBI’s Gene Expression Omnibus [GEO:GSE84500] (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE84500).
Statistical analysis
Student’s t test was used for statistical comparisons. Numeric data are represented as mean ± standard deviation of triplicate experiments, unless stated otherwise.