Differentiation of human bone marrow-derived MSCs into adipocytes
Frozen bone marrow mononuclear cells were purchased from Allcells (Allcells, Emeryville, CA, USA). After thawing, mononuclear cells were resuspended in an α-minimal essential medium (α-MEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated FBS (Invitrogen) and 1% antibiotic/antimycotic solution (Invitrogen). The cells were plated at a density of 1 to 5 × 106 cells per 100-cm2 dish. The cultures were maintained at 37°C in a 5% CO2 incubator and the medium was changed after 48 h and every 3 to 4 days thereafter. When the MSCs were confluent, the cells were recovered by the addition of 0.25% trypsin/ethylenediaminetetraacetic acid (EDTA) (Invitrogen). MSCs (passage 2 to 3) were plated in a 75-cm2 flask at a density of 1 to 2 × 104 cells and cultured in α-MEM with 10% FBS for 7 days. The medium was replaced with adipogenic medium, and the cells were cultured for an additional 14 days as described previously . Human MSCs, passage 3, were cultured in the presence of the HO-1 inducer cobalt protoporphyrin (CoPP) (5 μM) and with the HO activity inhibitor tin (Sn4+)-mesoporphyrin (SnMP) (5 μM), which were administered every 2 days.
HO activity measurement
Heme oxygenase activity was measured in hMSCs by carbon monoxide (CO) production in cellular homogenates. Briefly, hMSCs were homogenized in Sucrose (255 mM)-Tris hydrochloride (20 mM) buffer (pH 7.4) with NP-40 (1% w/v), EDTA (1 mM), phenylmethylsulfonyl fluoride (PMSF) (1 mM) and mammalian protease inhibitor cocktail (5% v/v). After homogenization, samples were centrifuged, at 6000 × g for 30 minutes at 4°C, and the supernatant collected for measurement of HO activity; 100 μg protein/sample was incubated, in gas-sealed vials, in Sucrose-Tris buffer along with nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) (1 mM) and excess heme (40 μM), in both the absence and the presence of SnMP (2 μM). Samples were incubated in a water bath, in the absence of light, at 37°C for 60 minutes, after which, the HO reaction was stopped by placing the samples in ice. CO generation was quantitated in the headspace using gas chromatography/mass spectrometry (GC/MS), as previously described , using C13O16 as an internal standard. Results are expressed as HO-dependent CO generation by subtracting the amount of CO in the presence of SnMP. CO generated is expressed as pmoles/mg protein/hour.
Effect of CoPP on adipogenesis
To measure the effect of increased HO-1 expression on MSC-derived adipocyte differentiation, cells were treated with 0.5, 1.0, 2.0, 5.0, and 10.0 μM of CoPP every 4 days. After 14 days, cells were stained with Oil Red O solution.
Oil Red O staining
Staining was performed using 0.21% Oil Red O in 100% isopropanol (Sigma-Aldrich, St. Louis, MO, USA). Briefly, adipocytes were fixed in 10% formaldehyde, stained with Oil Red O for 10 minutes, rinsed with 60% isopropanol (Sigma-Aldrich), and the Oil Red O eluted by adding 100% isopropanol for 10 minutes and the optical density (OD) measured at 490 nm, for 0.5 sec reading.
Measurement of lipid droplet size
After induction of adipogenesis, lipid droplets were stained with 2 μM boron-dipyrromethene (BODIPY) 493/503 (Molecular Probes, Eugene, OR, USA) . Cell size was measured using an ImagePro Analyzer (MediaCybernetics, Inc., Bethesda, MD, USA). The classification of the size of lipid droplets was based on size by area (pixels).
Cell viability test by lactic dehydrogenate assay (LDH)
We followed the manufacturer's protocol (LDH Assay kit, Cayman, Ann Arbor, MI, USA). Briefly, hMSC and adipocytes at day 14 were plated in 96-well plates for 1 day. Next day, cell layers were washed twice with PBS, and then cells were treated with various concentrations of CoPP (0 to 10 μM). After incubation for 24 h, and addition of 100 μl of reaction mixture to each well, cells were incubated for 4 hours at 37°C and 5% CO2 in a humidified incubator. Absorbance was measured in the 96-well microplate using a microplate reader at 490 nm with 650 nm as the reference wavelength, and the percentage of LDH release for each sample was normalized according to the absorbance reading from samples treated with 0.5% Triton X-100. All analyses were replicated eight times.
Cytokine array and adiponectin
TNFα and adiponectin (high molecular weight, HMW), were measured as previously described [25, 40] by Cytokine SearchLight Infrared arrays (Pierce Biotechnology, Inc., Woburn, MA, USA).
HO-1 siRNA transfection
Cells were treated with three different predesigned siRNAs of the HO-1 gene (SASI_ Hs01_00035068, SASI_Hs01_00035065 and SASI_Hs01_00035067 from Sigma-Aldrich, St. Louis, MO, USA). According to the manufacturer's protocol, adipogenic media containing siRNA using NTER (Sigma-Aldrich) was replaced every 48 h. Briefly, a nanoparticle solution was incubated with 10 nM siRNA. After 20 minutes cells were treated with siRNA solution during adipogenesis, which was halted after 10 days.
Measurement of MSC-derived adipocyte signaling molecules
Cells were maintained at -80°C until required for assay. Frozen cells were pulverized and placed in a homogenization buffer (10 mM phosphate buffer, 250 mM sucrose, 1 mM EDTA, 0.1 mM PMSF and 0.1% tergitol, pH 7.5). Homogenates were centrifuged at 27,000 × g for 10 minutes at 4°C. The supernatant was isolated and protein levels were assayed (Bradford Method). The supernatant was used for measurement of HO-1, Wnt10b, β-catenin, Pref-1, C/EBPα, Peg-1/Mest, pGSK3β, shh, PPARγ and β-actin levels as described previously [25, 41]. β-Actin was used to ensure adequate sample loading for all western blots.
Quantitative real-time PCR analysis
Total RNA was extracted from differentiated human mesenchymal stem cells using 5-Prime PerfectPure RNA Tissue Kit (Fisher Scientific Company, LLC, Wilmington, DE, USA). Total RNA was read on a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) and cDNA was synthesized using High Capacity cDNA Reverse-Transcription Kit (Life Technologies, Grand Island, NY, USA). PCR amplification of the cDNA was performed by quantitative real-time PCR using TrueAmp SYBR Green qPCR SuperMix (Smart Bioscience, Philadelphia, USA). The thermocycling of IL-8 and Secreted frizzled-related protein 1 (SFRP1) protocol consisted of 5 minutes at 95°C, 40 cycles of 15 sec at 95°C, and 30 sec at 60°C, and finished with a melting curve ranging from 60 to 95°C to allow distinction of specific products. Normalization was performed in separate reactions with primers to 18S mRNA (TTCGAACGTCTGCCCTATCAA and ATGGTAGGCACGGCGACTA).
Statistical significance (P < 0.05) of differences between experimental groups was determined by the Fisher method for analysis of multiple comparisons. For comparison between treatment groups, the null hypothesis was tested by either single-factor analysis of variance (ANOVA) for multiple groups, or the unpaired t-test for two groups, and the data are presented as mean ± standard error (SE).