Proteome Analysis of Rat Bone Marrow Mesenchymal Stem Cell

Aug 3, 2010 - Biotechnology Institute, Stem Cell Institute, Ankara, Turkey, and Gazi ... and the potential to differentiate in culture into several ce...
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Proteome Analysis of Rat Bone Marrow Mesenchymal Stem Cell Differentiation Betu ¨l C ¸ elebi,† A. Eser Elc¸in,†,‡ and Y. Murat Elc¸in*,† Tissue Engineering, Biomaterials and Nanobiotechnology Laboratory, Ankara University, Faculty of Science, Biotechnology Institute, Stem Cell Institute, Ankara, Turkey, and Gazi University, GEF, Division of Biology Education, Ankara, Turkey Received May 20, 2010

Bone marrow multipotent stromal cells (or mesenchymal stem cells; MSCs) have the capacity for renewal and the potential to differentiate in culture into several cell types including osteoblasts, chondrocytes, adipocytes, cardiomyocytes, and neurons. This study was designed to investigate the protein expression profiles of rat bone marrow MSCs during differentiation into adipogenic (by dexamethasone, isobutylmethylxanthine, insulin, and indomethacin), cardiomyogenic (by 5-azacytidine), chondrogenic (by ascorbic acid, insulin-transferrin-selenous acid, and transforming growth factor-β1), and osteogenic (by dexamethasone, β-glycerophosphate, and ascorbic acid) lineages by well-known differentiation inducers. Proteins extracted from differentiated MSCs were separated using two-dimensional gel electrophoresis (2-DE) and protein spots were detected using Sypro Ruby dye. Protein spots that were determined to be up- or down-regulated when the expression of corresponding spots (between weeks 1 and 2, 1 and 3, 1 and 4) showed an increase (g2-fold) or decrease (e0.5-fold) were successfully identified by MALDI-TOF-MS. In summary, 23 new proteins were identified either up- or down-regulated during differentiation experiments. Keywords: Mesenchymal stem cells • Bone marrow • Differentiation • Proteome analysis • MALDITOF-MS • Rat • Adipogenesis • Cardiomyogenesis • Chondrogenesis • Osteogenesis • Stem cells

Introduction Bone marrow (BM) multipotent stromal cells (or mesenchymal stem cells; MSCs) represent an alternative source for cellular therapies. These multipotent cells have the capacity for self-renewal1 and the tendency to differentiate into a variety of cell types, including osteoblasts, chondrocytes, adipocytes, cardiomyocytes, and neurons.2-8 Several studies have examined their potential to differentiate from the bone marrow (BM) where it provides a special source for MSCs. It is known that the expression of 8 common genes (the master control genes) of human (h) adult MSCS is increased during chondrogenic, adipogenic and osteogenic differentiation, as reported by Baksh et al.9 According to their findings, 235 genes are common during adipogenesis and osteogenesis in comparison to 3 during chondrogenesis and osteogenesis, and 10 genes during adipogenesis and chondrogenesis; thus designated two cell types (adipocytes and osteoblasts) that might come from a common precursor.9 Digirolamo et al. found that BM-MSCs lost their adipogenic differentiation after 12 passages and underwent osteogenesis by depositing calcium.10 The cellular processes and molecular characteristics of differentiating MSCs * To whom correspondence should be addressed: Ankara University, Faculty of Science, Department of Chemistry, Biochemistry Division, Degol Caddesi, Tandogan, 06100 Ankara, Turkey. Phone, +90(312) 212-6720; fax, +90(312) 223-2395; e-mail, [email protected]. † Ankara University. ‡ Gazi University. 10.1021/pr100506u

 2010 American Chemical Society

in subcultures and their progenies were elucidated at the gene level. Recently, it has been reported that differentially regulated passage-specific proteins may play a role in the decrease of rat MSC proliferation potential under serial subculturing.11 The possibility of rat BM-MSC differentiation in unstimulated culture conditions into other cell types in a slow and progressive way is pointed out.11 Mass spectroscopy (MS) based proteomics is becoming an efficient method for the detection of cellular processes at the protein level, allowing rapid identification of proteins during differentiation.12,13 For example, the effect of transforming growth factor-β (TGF-β) on the MSC proteome has been demonstrated by identification of about 30 altered proteins.14 Ye et al. investigated the effects of 5-azacytidine (5-aza) on rat BM-MSCs, which is a particular inducer of cardiomyogenic differentiation. They demonstrated that 9 proteins among 34 identified with MALDI-TOF-MS analysis showed distinct regulation in MSCs after 5-aza treatment.15 Sun et al. studied the proliferation and osteogenic potential of human MSCs during serial subculture and identified proteins which were differentially regulated during serial subculture and osteogenic differentiation using proteome analysis.16 By using two-dimensional (2D) gel electrophoresis, Welsh et al. highlighted some of the proteins down- or up-regulated during the eight daysexperimental period, known to be essential for adipogenesis, such as the cytoskeletal, signaling and cytokinesis proteins.17 Journal of Proteome Research 2010, 9, 5217–5227 5217 Published on Web 08/03/2010

research articles In the present work, our aim was to identify proteins secreted by the rat bone marrow mesenchymal stem cells at different steps of differentiation toward adipocytes, chondrocytes, osteocytes and cardiomyocytes. To compare protein expressions, we used 2-DE and MALDI-TOF-MS for rat MSCs at days 7, 14, 21, and 28 of the induced culture conditions.

Materials and Methods Rat BM-MSC Isolation and Culture. Rat bone marrow mesenchymal stem cells were isolated from 8-10 weeks old male Wistar rats weighing 150-200 g (n ) 12). All protocols involving animals were conducted according to the standards of international regulations. Rats were anesthetized by avertin injection. Under anesthesia, marrow was aspirated from the long bones (tibias and femurs) into Hank’s Buffered Salt Solution (HBSS, Biochrom AG, Berlin, Germany) containing 2% heparin (100 U/mL) by centrifugation. Low-density mononuclear cells were isolated by density gradient centrifugation in Polymorphoprep (Axis-Shield, Norton, MA) at 800g for 30 min at room temperature. Cells were plated in four parallel T-75 culture flasks (75 cm2), at 8.0 × 103 cells/cm2 density and cultured in R-Minimum Essential Medium (R-MEM, Gibco, Paisley, U.K.) supplemented with 20% fetal calf serum (FCS; Hyclone, Thermo, Rockford, IL), 2 mM L-glutamine, 100 U/mL penicillin, and 100 µL/mL streptomycin (all from Sigma, St.Louis, MO) at 37 °C humidified atmosphere containing 5% CO2 for 3 days before the first medium change. Nonadherent cells were removed and the medium was exchanged with fresh medium twice a week. At 80-85% confluence, cells were trypsinized with 0.25% trypsin-EDTA (Sigma). Cells from Passage 1 and 2 were used in differentiation experiments. MSCs of separate donors were pooled (n ) 3) and plated at an initial cell density of 4.0 × 102 cells/cm2, into four T-25 flasks (25 cm2) and four 13 mm-coverslips (Thermanox, Nunc, Naperville, IL), in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Paisley, U.K.) supplemented with 10% FCS (Hyclone), 2 mM L-glutamine, 100 U/mL penicillin, and 100 µL/mL streptomycin (Sigma) (basal medium) (n ) 4). After a week of cell expansion, differentiation agents were added into corresponding culture flasks. Experimental groups were constituted by modifying the basal medium with the corresponding differentiation inducers as follows: (i) for osteogenic differentiation, 10-8 M dexamethasone, 10 mM β-glycerophosphate, and 50 mg/mL ascorbic acid (all from Sigma); (ii) for chondrogenic differentiation, 1% insulin-transferrin-selenous acid combination (ITS), 5 ng/mL TGF-β1, 1% nonessential amino acid stock solution, 50 µg/mL ascorbic acid (all from Sigma); (iii) for adipogenic differentiation, 10 mM indomethacin, 0.5 mM 3-isobuthylmethyl-xanthine (all from Fluka, Buchs, Germany), 1 µM dexamethasone, 10 µg/ mL insulin (all from Sigma); (iv) for cardiomyogenic differentiation, 10 µM 5-azacytidine (Sigma). While the first three groups were kept under the defined conditions for a duration of 28 days, the cardiomyogenic differentiation group was treated with 5-aza for the first 24 h, and then the medium was switched to the basal medium in the following days up to 28 days. All corresponding media were changed twice a week. The phase contrast images of differentiating rat MSC morphology were regularly collected by using a Nikon TS 100 inverted microscope with 20× objective and a Nikon digital CCD camera. Differentiating MSC cultures were collected as cell samples for proteomic analysis. Immunohistochemistry (IHC) and Histochemical Analysis. At the end of the first, second, third and fourth weeks, coverslips 5218

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C ¸ elebi et al. containing MSCs cultured on osteogenic, adipogenic, chondrogenic, and cardiomyogenic induction were washed with phosphate-buffered saline (PBS), then were fixed in ice-cold methanol for 10 min, and only for adipogenic induced cells on coverslips, samples were fixed in 10% formalin for 30 min and then air-dried. Osteo-induced specimens were permeabilized with distilled (d) H2O, then endogenous peroxidase activity was blocked with peroxidase block (LabVision, Fremont, CA, ABD) for 10-15 min. After washing with dH2O, coverslips were incubated with Ultra V Blok (LabVision). To minimize nonspecific binding, 2% blocking serum containing PBS was applied for 10-15 min, and then the specimens were washed again with PBS and were incubated with primary antibodies for an hour at room temperature, or overnight at 4 °C. Immunohistochemical detection of adipogenic differentiation was carried out using mouse monoclonal anti-PPARγ IgG1 (1:50) (Santa Cruz Biotechnology, Santa Cruz, CA). Immunohistochemical detection of cardiomyogenic differentiation was carried out using goat polyclonal anti-cardiac troponin-T IgG (1:50) (Santa Cruz Biotechnology). Immunohistochemical detection of osteogenic differentiation was carried out using mouse monoclonal anti-osteonectin (OSN) IgG (1:50) (Alexis, San Diego, CA), goat polyclonal anti-osteopontin (OSP) IgG (1:100) (Santa Cruz Biotechnology), and goat polyclonal antiOSC IgG (1:100) (Santa Cruz Biotechnology) primary antibodies. Species- and isotype-specific secondary biotinylated goat antimouse IgG (1:100) and rabbit anti-goat IgG (1:100) (Zymed) antibodies were used to bind the primary antibodies. Enzymatic reaction was carried out with streptavidin horseradish peroxidase, and AEC (Lab Vision) chromogen was used to develop the red staining. Alcian blue and hematoxylene-eosin histologic stainings were performed to demonstrate chondrogenic and cardiomyogenic differentiated samples at days 14 and 28. The samples were visualized under a Leica DM 4000B model digital microscope (Wetzlar, Germany). Preparation of Protein Samples. For proteomic analysis, at predetermined time points, four flasks which were left to osteogenic, chondrogenic, adipogenic, and cardiomyogenic differentiation were washed with ice-cold PBS solution for at least three times. Then, the cells were removed from the cultures using a cell scraper (Orange Scientific, Braine-l’Alleud, Belgium), and lysed in the lysis buffer containing 0.5% TritonX100 (Sigma), 50 mM Tris HCl (pH 7.5-8), 10 mM tris (2carboxyethyl) phosphine (TCEP; Pierce, Thermo), complete protease inhibitor (Roche, Mannheim, Germany), and sonicated for 30 s. The samples were centrifuged at 18 000g for 10 min to remove any insoluble cell debris. Protein contents in the cell lysates were determined by the Bradford method (Bio-Rad, Hercules, CA) and the remaining protein solutions were stored at -80 °C for further proteomic analysis. Protein Separation and Image Analysis. Extracted proteins were precipitated with trichloroacetic acid (TCA). One hundred microliters of 10% TCA was mixed with 1000 µg of soluble protein, followed by incubation on ice for 15 min, then centrifuged at 18 000g for 10 min, and washed two times with 75% acetone by a dilution of 1:1 (w/w). After centrifugation of 5 min at 18 000g, the pellet was ready for the next step. Two dimensional electrophoresis (2-DE) experiments were carried out according to the manufacturer’s instructions (Bio-Rad). For the first dimension, 300 µL of rehydration buffer consisting of 7 M urea (Bio-Rad), 2 M thiourea (Sigma), 1% DTT (Fermentas, Glen Burnie, MD), 4% CHAPS (Sigma), 0.5% (v/v) IPG buffer (Fluka, Buchs, Germany) containing 200 µg of proteins was

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Proteomics of Rat MSC Differentiation Table 1. Medium Types with Inducers and Days of Induction type of medium

Basal medium

Adipogenic Osteogenic Chondrogenic Cardiomyogenic

inducers

days of induction

Dulbecco’s Modified Eagle’s Medium with 10% fetal calf serum, 100 U/mL penicillin and 100 µL/mL streptomycin, and 2 mM L-glutamine 10 mM indomethacin, 0.5 mM 3-isobuthylmethyl-xanthine, 1 µM dexamethasone, 10 µg/mL insulin 10-8 M dexamethasone, 10 mM β-glycerophosphate, 50 mg/mL ascorbic acid 1% insulin-transferrin-selenous acid combination, 5 ng/mL TGF-β1, 1% nonessential amino acids, 50 µg/mL ascorbic acid 10 µM 5-azacytidine

subjected to isoelectric focusing (IEF) on an IPG strip 17 cm, pH 3-10 linear gradient (Bio-Rad) at 20 °C.18 IPG strips were actively rehydrated with the sample mixture at 50 V for 15-16 h to enchance protein uptake. With Protean IEF Cell (Bio-Rad), proteins were focused using the following protocol: 250 V for 15 min, the second 10 kV for 3 h at rapid ramping mode, and the final phase at 10 kV for 60 min. All IEF steps were carried out at 20 °C. Before carrying out 2D sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), IPG strips were placed in an equilibration buffer consisting of 6 M urea (BioRad), 30% glycerol (Bio-Rad), 2% SDS (Sigma), 50 mM Tris HCl (pH 8.8), and 2% DTT for 15 min while being shaken. The strips were then transferred to the same equilibration buffer in which DTT was replaced with 2.5% iodoacetamide (Sigma) and shaken for a further 15 min. The strips were then transferred onto the second dimensional 1.5 mm thick 4% stacking and 10% running polyacrylamide gels and sealed in place with 0.5% low-melting point agarose (Sigma). Separation in the second dimension was carried out using BioRad Protean Plus Dodeca Cell system (20 cm ×25 cm, Bio-Rad) at 30 mA per gel until the bromophenol blue dye marker had reached the bottom of the gel. The temperature of the electrophoresis system was kept at the range of 11-12 °C. The protein spots of the protein extracts were visualized by Sypro Ruby dye according to the protocol described by Sigma. Spot detection and matching was automatically performed by using PDQuest 7.2 software (Bio-Rad). Proteins were determined to be up- or down-regulated, when the expression of the corresponding spots (1-2, 1-3, 1-4) showed g2-fold increase or e0.5-fold decrease. In-Gel Digestion of Proteins Separated by 2D-Gel Electrophoresis. To identify proteins, the spots were excised from the stained gels via BioRad’s Spot Cutter. Each selected spots was cut from the gels into small pieces (1 mm2), placed into 96 microplate wells filled with dd-H2O. For trypsinization, first gel fragments were swollen in 10 mM DTT (Fermentas) in 0.1 mM NH4CO3 (Sigma) and incubated for 30 min at 37 °C to reduce the protein, then gels were shrunk using acetonitrile (Sigma). After removing the supernatant, 55 mM iodoacetamide (Sigma) and 0.1 mM NH4CO3 (Sigma) were added. Then, the gels were washed with 0.1 mM NH4CO3 for 15 min and dried with acetonitrile. Rehydration of gel particles was carried out in a digestion buffer of 50 mM NH4CO3 containing 12.5 ng/µL trypsin (Promega) at 4 °C for 30-45 min. For gel digestion, 15 µL of NH4CO3 without trypsin was added onto each gel. As the last step, gels were incubated at 37 °C for 16 h. A solution of 50% acetonitrile/5% formic acid (ACN, Riedel de Hae¨n, Seelze, Germany) was added to the digest mixture to recover the peptides and sonicated for 10 min. The last step was repeated for at least three times; each fraction was collected in another well and then lyophilized.

-

0-28 0-28 0-28 0-1

Protein Identification by MALDI-TOF-MS. Protein identification was achieved by peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) (Waters Corporation, Milford, MA). The dried extracts were reconstituted with matrix solution composed of 10 mg of R-cyano-4-hydroxycinnamic acid (recrystallized CHCA, Sigma) in 1 mL of 49.5% acetonitrile, 49.5% ethanol (Sigma), and 1% of 0.1% TFA (Sigma). Then, 1.5 µL of dissolved sample was loaded onto a MALDI plate. After airdrying, digested peptides were analyzed using MALDI-TOFMS. The following mass search parameters were set: peptide mass tolerance, 50 ppm; mass spectra over the m/z range 800-3000 Da, allowance of missed cleavage, 1; consideration for variable modifications such as oxidation of methionine and carbamidomethyl of cysteines. The spectrometer was run with the following settings: source voltage, 15 kV; pulse voltage 3 kV; MCP detector voltage, 1850 V; reflectron voltage, 500 V; TLF delay 500 ns. Spectra were internally calibrated using trypsinized alcohol dehydrogenase (147 kDa; Sigma). Glu-Fibrinopeptide B (1570 Da; Sigma) was used as the external standard. Proteins were identified by their peptide mass fingerprint (PMF) with Protein Lynx Global Server (PLGS) and Swiss-Prot (http:// www.expasy.ch).

Results and Discussion Although the effects of varying differentiation cocktails on the MSCs have been widely studied, characterization of their protein profiles following differentiation during long-term cultures/passages has not been clearly performed. In the present study, rat bone marrow MSCs were induced to differentiate into adipogenic, osteogenic, chondrogenic, and cardiomyogenic lineages. Types of media used for specific inductions and durations of application are summarized in Table 1. The changes of expression levels of proteins during MSC differentiation were monitored by 2-DE during four passages (within 28 days). Immunohistochemical and histochemical analysis were applied to evaluate the expression of differentiation markers of induced MSC cultures. Plasticity and Differentiation Potential of rMSCs during Subcultures. Mesenchymal stem cells were isolated from the rat bone marrow by Polymorphoprep gradient centrifugation technique. MSCs were attached to the culture plate and proliferated in the culture medium. The nonadherent cells of the marrow were washed away during culture medium changes. The expression of specific differentiation markers were evaluated for a duration of 28 days. Adipogenic differentiation implemented by dexamethasone, IBMX, insulin, and indomethacin showed lipid droplet formation and decrease in cell content between 7 and 14 days of culture (both fibroblast-like cells and adipogenic cells) (Figure 1B,C). After 14 days, rMSCs were Journal of Proteome Research • Vol. 9, No. 10, 2010 5219

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Figure 1. Adipogenic differentiation of rat MSCs: (A) 7th day of culture; (B) 14th day of culture demonstrating the formation of lipid droplets; (C) decrease of cell content (fibroblast like cells and adipogenic cells) (A-C; phase contrast; Hoffman modulation); (D) Anti-PPAR-γ dye. Adipogenic conditions: 1 µM dexamethasone, 0.5 mM 3-IBMX, 10 µg/mL insulin, 10 mM indomethacin. (Bars ) 100 µm).

Figure 2. Cardiomyogenic differentiation of rat MSCs: (A) 7th day of culture; (B) 14th day of culture, cell morphology change insignificant; (C, D) 28th day of culture, cardiomyocyte like morphology observed (arrows); (A-D, phase contrast; Hoffman modulation); (E) 28th day of culture, H&E-staining; (F) 28th day of culture, anticardiac Troponin-T staining. Cardiomyogenic inducer: 10 µM 5-azacytidine. (Bars ) 100 µm).

positive to anti-PPAR-γ staining (Figure 1D). Cardiomyogenic differentiation was performed using 24 h 5-azacytidine treatment. Cell morphology did not change during the first week (Figure 2B) with similarity to control cultures. However, cardiomyocyte-like morphology could be observed between days 14 and 21 (Figure 2C,D); hematoxylene-eosin and anticardiac 5220

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Figure 3. Chondrogenic differentiation of rat MSCs: (A) 7th day of culture; (B) 14th day of culture (phase contrast; Hoffman modulation); (C) 14th day of culture, Alcian blue staining. Chondrogenic inducers: 1% ITS, 5 ng/mL TGF-β1, 50 µg/mL ascorbic acid. (Bars ) 100 µm).

Troponin-T stainings at day 28 confirmed the cardiomyogenic differentiation of rMSCS (Figure 2E,F). Chondrogenic differentiation (by using ascorbic acid, ITS, and TGF-β1) of rMSCS could be shown as well. Cell morphology at the seventh and 14th days of culture is shown in Figure 3A,B. Alcian bluestaining of the 14th day of chondrogenic culture is presented in Figure 3C. While micropellet systems are usually preferred for chondrogenic differentiation experiments, a certain level of Alcian blue staining could be observed for standard confluent rMSCs. Osteogenic differentiation was obtained using dexamethasone, β-glycerophosphate, and ascorbic acid as inducers (Figure 4). While rMSCs showed adipocyte-like morphology during the first week of culture (Figure 4A), they turned into a cubical morphology between 7 and 14 days of culture (Figure 4B,C). At the third week of osteogenic culture, rMSCS started to form three-dimensional structures and mineral accumulation could be observed (Figure 4D). rMSCs under osteogenic culture conditions were positively stained with anti-Osteonectin at the second week, anti-Osteopontin at the third week, and anti-Osteocalcin at fourth culture week, as shown in Figure 4E-G. Proteome Analysis. The 2DE gel analysis was performed on 17-cm Bio-Rad strips, wide pH range 3-10 in the first dimension, and 10% SDS-PAGE in the second dimension. The representative 2DE gels of the adipogenic, cardiomyogenic, chondrogenic, and osteogenic cultures of rMSCs at days 7, 14, 21, and 28 are presented in Figures 5-8. By using the Bio Rad PDQuest 7.2 software, we analyzed the initial culture condition (after 7 days of induction with differentiation cocktails) comparing with that of the second, third, and fourth weeks and obtained protein spots which were determined to be up- or down-regulated when the expression of corresponding spots (1-2, 1-3, 1-4) showed an increase (g2-fold) or decrease (e0.5-fold). During the adipogenic differentiation, 184, 219, and 171 protein spots were designated to be up- or down-regulated as a result of 1-2, 1-3, 1-4 comparative conditions, respectively, and 43 intersected spots were chosen for protein identification. During the cardiomyogenic differentiation, 110, 170, and 74 protein spots were designated to be up- or downregulated as a result of 1-2, 1-3, 1-4 comparative conditions,

Proteomics of Rat MSC Differentiation

research articles glands, pancreas, kidneys, placenta, spleen, lungs, ovaries, testes, and brain, in addition of rat heart and skins.19,20 The apo D mRNA is expressed primarily by fibroblasts. Provost et al. indicated that apo D mRNA was detectable only in nonproliferating quiescent and senescent cultures and also its expression was independent of exogenous cholesterol.21 It is shown that the addition of dexamethasone to cultures induced the expression of apo D and reduced cell proliferation.22 Although, apo D is lower in MSCs than in fibroblasts, its expression increased after MSC-derived osteogenic differentiation.23 According to Cha et al., apo D and fatty acid binding protein (FABP) mRNA expressions were increased by over 9-fold in differentiated adipocytes compared with preadipocytes.24 We also found an increase in the expression of apo D during adipogenic differentiation.

Figure 4. Osteogenic differentiation of rat MSCs: (A) MSCs showed adipo-like morphology at the seventh day of culture; (B and C) cuboidal cell morphology and 3D-cell aggregation started to appear at the 7-14th day period; (D) bone-like mineralization started to appear at the 21st day of culture (phase contrast; Hoffman modulation). Immunohistochemical stainings: (E) AntiOsteonectin, at week 2; (F) Anti-Osteopontin, at week 3; (G) AntiOsteocalcin at week 4. Osteogenic inducers: 50 mg/mL ascorbic acid, 10 mM β-glycerophosphate, 10-8 M dexamethasone. (Bars ) 100 µm).

respectively, and 26 intersected spots were chosen for protein identification. During the chondrogenic differentiation, 214, 37, and 0 protein spots were designated to be up- or downregulated as a result of 1-2, 1-3, 1-4 comparative conditions, respectively, and 14 intersected spots were chosen for protein identification. During the osteogenic differentiation, 215, 230, and 183 protein spots were designated to be up- or downregulated as a result of 1-2, 1-3, 1-4 comparative conditions, respectively, and 13 intersected spots were chosen for protein identification. Protein Identification by Mass Spectrometry. The protein spots in different sets of gels were identified by using the MALDI-TOF-MS on the basis of peptides molecular weights, isoelectrical points, sequence coverages, and comparison between observed to theoretical masses in tryptic digests of all known proteins from Swiss-Prot databases. Analyses of the selected spots yielded 23 protein data given in Table 2. Function of Identified Proteins. 1. Adipogenic Differentiated Proteins. Apolipoprotein D precursor, ATP synthase lipid binding protein mitochondrial precursor, Neurotrimin precursor, A kinase anchor protein 8, Pannexin 1, Proteoglycan link protein1 precursor, Sushi repeat containing protein SRPX precursor, and ADAM 15 precursor were identified among 9 intersected proteins that were up- and down-regulated during adipogenic differentiation. Apolipoprotein D (apo D) precursor is a member of the lipocalin family of transporters. It is poorly expressed in the human liver and intestine, but highly expressed in the adrenal

ATP synthase lipid binding protein mitochondrial precursor is anchored in the inner membrane of the organelle by an intrinsic membrane sector Fo.25 After cellular exposure to insulin, lipogenesis is activated in adipocytes and many enzymes are highly up-regulated and activated in adipocytes. We found an increase in the expression of ATP sythase during adipocyte differentiation. Kim et al. highlighted that ATP synthase complex was found not only in the mitochondria, but also on the cell surface of adipocytes and expression of this enzyme and mitochondrial biogenesis were highly increased during adipogenesis. They also demonstrated that cytochorome c along with ATP synthase were highly expressed in adipocytes that were active for oxidative respiration to generate NADH and ATP.26 Neurotrimin precursor (Ntm) is a neural adhesion protein. Krizsan-Agbas et al. have shown that there was an upregulation of Ntm expression during the first 6 h and a downregulation up to 24 h, after supplementing smooth muscle cells cultures with steroid hormones.27 Adipose stem cells are capable of undergoing neural differentiation as reported by Bunnell et al.28 After induction of adipose stem cells with indomethacin, insulin, and IBMX, murine, nonhuman primate, and human cells have also been found to express additional neuronal markers.29,30 We observed a down-regulation of Neurotrimin during our adipogenic differentiation experiments. A kinase anchor protein 8 is an anchoring protein that mediates the subcellular compartmentation of cAMP-dependent protein kinase (PKA type II). This protein is down-regulated during adipogenic differentiation. Proteoglycan link protein1 precursor stabilizes the aggregates of proteoglycan monomers with hyaluronic acid in the extracellular cartilage matrix. No¨th et al. have highlighted that MSCs derived from human trabecular bone underwent adipogenesis in vitro and chondrogenesis in vivo.31 TGF-β1 treated cells expressed cartilaginous matrix components such as collagen II and proteoglycans link protein.31 Wnt/β-catenin is an important signaling pathway for osteogenic, adipogenic, and chondrogenic differentiation. It has been shown that Wnt/βcatenin signaling inhibits adipogenic and enhances chondrogenic differentiation of pericytes.32 Proteoglycan link protein1 precursor is down-regulated during adipogenic differentiation. Pannexin (Panx) 1: gap junctions are composed of proteins that form a channel connecting the cytoplasm of adjacent cells. Three families of gap junction proteins are connexins, innexins, and pannexins. Panx appear to be suited for ATP and calcium (Ca2+) homeostasis by mechanisms implicating both gap junction and nonjunctional functions.33 Panx1 has Journal of Proteome Research • Vol. 9, No. 10, 2010 5221

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Figure 5. 2-DE gel analysis of proteins extracted from adipogenic differentiated rat BM-MSCs at four time points. The first dimension was performed using 200 µg of total soluble proteins on linear gradient IPG strips, pH 3-10. In the second dimension, 10% SDS-PAGE gels were used, and the proteins were visualized using Sypro Ruby.

Figure 6. 2-DE gel analysis of proteins extracted from cardiomyogenic differentiated rat BM-MSCs at four time points. The first dimension was performed using 200 µg of total soluble proteins on linear gradient IPG strips, pH 3-10. In the second dimension, 10% SDS-PAGE gels were used, and the proteins were visualized using Sypro Ruby.

been proposed to provide a paracrine signaling pathway by forming membrane channels that are permable to ATP.34 It has been implicated in the leakage of Ca2+ from endoplasmic reticulum and has a role of tumor suppressor.35,36 Panx1 was down-regulated during our adipogenic differentiation experiments. 5222

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ADAM 15 precursor is a member of the type I integral membrane proteins that contain disintegrin and metalloprotease domain. Herren et al. have shown its role in increasing cell adhesion, enhancing cell-cell interactions and decreasing cell migration.37 In osteoarthritis, ADAM15 is up-regulated in chondrocytes at the early stage of cartilage degeneration and

Proteomics of Rat MSC Differentiation

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Figure 7. 2-DE gel analysis of proteins extracted from chondrogenic differentiated rat BM-MSCs at four time points. The first dimension was performed using 200 µg of total soluble proteins on linear gradient IPG strips, pH 3-10. In the second dimension, 10% SDS-PAGE gels were used, and the proteins were visualized using Sypro Ruby.

Figure 8. 2-DE gel analysis of proteins extracted from osteogenic differentiated rat BM-MSCs at four time points. The first dimension was performed using 200 µg of total soluble proteins on linear gradient IPG strips, pH 3-10. In the second dimension, 10% SDS-PAGE gels were used, and the proteins were visualized using Sypro Ruby.

enhances integrin-mediated chondrocyte adhesion to the collagen matrix.38 ADAM 15 precursor was also down-regulated in our adipocyte differentiation cultures. Sushi repeat containing protein SRPX precursor: URB which belongs to the sushi repeat containing protein superfamily is down-regulated during osteogenic differentiation of MSCs comparing to nondifferentiated ones. It has been shown that a 2 week long osteogenic induction results in a decrease and

finally disappearance of the URB expression.39 However, due to its up-regulation in the adipose tissue of obese bombesin receptor subtype 3 deficient mice, it was suggested to play a role in the regulation of body weight and energy metabolism.40 Sushi repeat containing protein precursor was upregulated in our adipogenic differentiation cultures. 2. Cardiomyogenic Differentiated Proteins. Among the six intersected proteins that were identified during cardiomyoJournal of Proteome Research • Vol. 9, No. 10, 2010 5223

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Table 2. Identities of Differentially Expressed Proteins in Rat Bone Marrow MSCs Following Adipogenic, Cardiomyogenic, Chondrogenic and Osteogenic Differentiationa differentiation

protein identity

Adipogenic

ADAM 15 precursor Apolipoprotein D precursor ATP synthase lipid binding protein mitochondrial Neurotrimin precursor A kinase anchor protein 8 Proteoglycan like protein precursor cartillage Pannexin Sushi repeat containing protein SRPX precursor Unknown protein Cardiomyogenic Homeobox protein Hox B7 Fatty acid binding protein epidermal ADP ribosylarginine hydrolase Tumor necrosis factor receptor superfamily member Equilibrative nucleoside transporter 2 Unknown protein Chondrogenic Leukemia inhibitory factor precursor LIF Cholin Collagen alpha 1 II chain precursor fragment Vascular cell adhesion protein 1 precursor GTP binding protein RAD Osteogenic Branched chain amino acid aminotransferase mitoc. 3 hydroxy 3 methylglutaryl coenzyme A reductase Unknown protein Unknown protein

accession number

theoretical Mr/pI

observed Mr/pI

Q9QYV0 P23593 Q06646 Q62718 Q63014 P03994 P60570 Q63769

88052/5.25 21635/4.7 14907/5.04 37998/5.62 76162/5.03 40262/7.98 48073/6.34 51560/8.9 120868/5.7 24243/7.64 15059/6.95 40042/5.4 29875/7.9 50265/6.3 45927/9.57 22113/8.99 134570/9.03 81246/5.11 33038/8.98 44276/7.19 96688/6.1 34547/6.31 100865/5.05

87995/5.3 21620/4.7 14907/10.2 37974/7.5 76114/4.8 40139/7.1 48221/7.1 51526/8.6 120762/5.7 23897/8.8 15064/7.7 39961/5.62 29895/8.39 50140/5.7 42257/9.6 22098/9.2 12011/9.9 81194/5.0 29035/6.7 30770/8.5 98232/6.4 34524/6.3 100802/4.9

P18864 P55053 Q02589 P15725 O54699 P17777 P05539 P29534 P55043 O35854 P14773

sequence matched PLGS coverage peptides score regulation

1.3 8.5 7.1 4.9 11.2 8.2 11.7 1.5 16.4 4.1 7.4 2.5 3.0 2.0 7.6 7.9 12.3 5.0 3.4 3.6 1.9 6.6 2.6

1 1 1 2 1 3 5 1 14 1 1 1 1 1 2 1 1 3 1 2 2 2 2

7.6 5.0 3.8 3.2 4.5 3.6 3.3 5.5

e0.5 g2.0 g2.0 e0.5 e0.5 e0.5 e0.5 g2.0

8.8 5.6 7.2 5.8 6.0

g2.0 e0.5 g2.0 e0.5 g2.0

6.4 5.5 4.8 7.0 6.9 5.5

e0.5 g2.0 g2.0 e0.5 e0.5 e0.5

a Observed Mr/pI values are based on the 2-DE migration, and the theoretical values were calculated from amino acid sequences. The protein samples were subjected to 2-DE, followed by Sypro Ruby staining and image analysis. Results were quantified from three gel sets. The spots of interest were excised from the gels and digested with trypsin. The resulting peptides were used in MALDI TOF-MS analysis, and the proteins were identified by searching the Swiss-Prot database using peptide sequences.

genic differentiation from 2DE were Fatty acid binding protein, Epidermal Homeobox protein Hox B7, Equilibrative nucleoside transporter 2, ADP ribosylarginine hydrolase, and Tumor necrosis factor receptor superfamily member 4. Homeobox protein Hox B7: homeobox genes have a regulatory function in differentiation processes ongoing in adult animal, including hematopoiesis. Human bone marrow cells were found to express Hox B7 in response to granulocytemacrophage CSF. It has been shown that Hox B7 is expressed only after stimulation with vitamin D3 which results in monocytic differentiation.41 Phinney et al. reported that HoxB7 and Hoxb4 were highly expressed by D3 and IDmMSCs which reflects a role in supporting proliferation, and self-renewal. Hox B7 also induces expression of FGF2 which functions in an autocrin manner to inhibit cellular differentiation.42 On the other hand, Care et al. demonstrated that Hox B7 induced an increase of the number and differentiation of granulo monocytic hematopoietic cells.43 According to our findings, there is an up-regulation of Hox B7 protein during the cardiomyogenic differentiation. Fatty acid binding protein epidermal (FABP5) plays a crucial role in the intracellular fatty acid transport by binding and properly targeting long chain fatty acid to their correct metabolic sites. The epidermal type (FABP5) is expressed in the adipose tissue. The heart/muscle type FABP3 has also been identified in the adipose tissue.44 FABPS and PPARs can also interact through direct protein-protein interactions either in a ligand dependent or in a ligand independent manner to activate the transcription of PPAR target genes. During adipogenesis, FABP3 and FABP5 showed an increase in expression.45 Our results showed that FABP5 was down-regulated during cardiomyogenic differentiation. Equilibrative nucleoside transporter 2: purine nucleotides (such as ATP) and their metabolites are physiologically essential 5224

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molecules in the heart. In cardiomyocytes, equilibrative nucleoside transporters (ENTs) facilitate the movement of purine nucleosides down their exogenous concentration gradient, and are responsible for the flux of adenosine across the plasma membrane.46 ENT2 is highly expressed in the human and mouse heart tissue and also in the skeletal muscle and plays a major role in mediating cardioprotectin.47,48 Our results showed that ENT2 was up-regulated during cardiomyogenic differentiation. ADP ribosylarginine hydrolase: ADP ribosylation is a reversible process in which NAD:arginine ADP-ribosyltransferases catalyze the forward reaction and ADP-ribosylarginine hydrolases remove the ADP-ribose moiety regenerating free arginine.49 In animals, ADP-ribosylarginine hydrolase activity was initially detected in the mouse fibroblasts where the arginine bond of the ADP-ribosylated histone substrate was cleaved.50 ADP ribosylarginine hydrolase was up-regulated during cardiomyogenic differentiation experiments. Tumor necrosis factor receptor superfamily member 4: Florholmen et al. explained that LIF-stimulated cardiomyoctes have an increased cardiac gene expression like the tumor necrosis factor and interleukin 4.51 Single nucleotide polymorphisms in the tumor necrosis factor (ligand) superfamily, member 4 gene, encoding Ox40L, have been associated with increased risk of myocardial infarction in women.52 Tumor necrosis factor receptor was down-regulated during our cardiomyogenic differentiation experiments. 3. Chondrogenic Differentiated Proteins. Four intersected proteins that were identified during chondrogenic differentiation from 2DE were Collagen alpha 1 (II) chain precursor, Vascular cell adhesion protein 1 precursor, GTP binding protein RAD, and Leukemia inhibitory factor precursor LIF Cholin. Leukemia inhibitory factor precursor LIF Cholin is a member of the interleukin 6 family and a potent stimulator of the gp130.53 Its activities include the induction of hematopoietic

Proteomics of Rat MSC Differentiation differentiation in normal and myeloid leukemia cells, and the induction of neuronal cell differentiation. Florholmen et al. explained that heart failure patients have an increased cardiac gene expression of leukemia inhibitory factor.53 In LIFstimulated cultures, tumor necrosis factor, interleukin 4, neurotensin, and somatostatin were up-regulated but it reduced the expression of ATP synthase complex, epidermal fatty acid binding protein, and insulin growth factor binding proteins 1 and 6.51 Leukemia inhibitory factor precursor was downregulated during our chondrogenic differentiation experiments. Collagen alpha 1 (II) chain precursor: the cartilage extracellular matrix contains an insoluble network of collagen IIcontaining fibrils that are integrated within an abundant anionic network of aggrecan and hyaluronan aggregates. As reported by Wilson et al. using proteomic approaches there is high expression of collagen alpha 1 (II) in cartilage tissues.54 Mouse bone marrow chondrogenically differentiated with TGFβ1 and insulin growth factor I have shown an increase in collagen type II expression.55 Collagen alpha chain precursor was up-regulated during our chondrogenic differentiation experiments. Vascular cell adhesion protein (V-CAM) 1 precursor appears to function in leukocyte-endothelial cell adhesion. It interacts with the β-1 integrin VLA4 on leukocytes, and mediates both adhesion and signal transduction. Kienzle et al. investigated the expression of adhesion molecules belonging to the immunoglobulin superfamily on human primary articular chondrocytes and found that VCAM-1, ICAM-1, and VLA-2 were constitutively expressed by human articular chondrocytes.56 IL1β/TNF-R and TGF-β antagonistically modulate the expression of adhesion molecules. VCAM-1 and ICAM-1 contribute to adhesion of T lymphocytes to chondrocytes, and thus may participate in host defense mechanisms during inflammatory joint conditions, such as the rheumatoid arthritis and after cartilage transplantation.56 Our results also demonstrated the up-regulation of Vascular cell adhesion protein during chondrogenic differentiation. GTP binding protein RAD is a Ras-like GTPase which is highly expressed in the heart, lung, and skeletal muscle. It associates with the actin-binding protein, β-tropomyosin and may participate in the regulation of the cytoskeleton.57 As reported by Moyers et al., overexpression of Rad inhibits insulin-stimulated uptake in rat myotubes and adipocytes.58 GTP-binding protein RAD was down-regulated during our chondrogenic differentiation experiments. 4. Osteogenic Differentiated Proteins. Among the four intersected proteins identified during osteogenic differentiation from 2DE were branched chain amino acid aminotransferase mitochondrial precursor, and 3-hydroxy-3-methylglutaryl coenzyme A reductase. Branched chain amino acid aminotransferase (BCAT) mitochondrial precursor catalyzes the reversible transamination of leucine, valine, and isoleucine.59 It indicates that enzyme activity is distributed between the cytosol and the mitochondrial compartment. Rat heart BCAT is solely mitochondrial.60 BCAT was down-regulated during our osteogenic differentiation experiments. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase catalyzes the rate-limiting step in the cholesterol biosynthesis.61 It has been shown that HMG CoA reductase inhibitors (statins) stimulate bone formation by increasing the expression of the bone morphogenetic protein 2 in the bone cells.62 HMG CoA was down-regulated during osteogenic differentiation.

research articles In this study, we observed the differentiation of BM-MSCs into four different lineages, in the light of morphological changes, by the specific differentiation markers (namely, antiPPAR-γ, anticardiac Troponin-T, anti-Osteonectin, anti-Osteopontin, anti-Osteocalcin, and Alcian blue dye), and by the protein expression characteristics. When the expression findings are considered, a high protein expression level was observed in some of the gels representing day 14; however, the mostly up- or down-regulated protein phase was seen at day 21, except for the chondrogenic differentiation group. As reported by Chiellini et al., adiponectin secretion and alkaline phosphatase activity of BM-MSCs increased in ongoing cultures, when differentiated into adipocytes and osteoblasts, respectively.63 Kulterer et al. have shown that hBM-MSCs differentiating into osteoblasts proliferated between days 0 and 4, entered the matrix maturation phase between days 4 and 14, and the mineralization phase started between days 14 and 21 under the osteogenic culture conditions.64 The osteogenic differentiation capacity of UCB-MSCs were reported by Kim et al.,65 where they stated the proteomic differentiation between days 7 and 21 and highlighted that the metabolic activity proteins were up- and down-regulated between these designated days. Also shown in our gel figures attained under similar conditions, these findings reflect the high metabolic or synthetic rate of the differentiating MSC cultures between days 14 and 21. Our findings demonstrated that the protein up- or downregulation of MSCs differentiating into the chondrogenic lineage slowed down after 14 days of culture, which stagnated even further after 21 days in terms of protein changes. It is known that chondrocyte monolayer cultures gradually lose their differentiated phenotype and may not form cartilage tissue. Kang et al.66 investigated whether chondrocytes cultured through various passages maintain their potential to reexpress a chondrogenic phenotype. Passage 2 chondrocytes exhibited a high expression of collagen type II and a low expression of collagen type I. In contrast, passage 5 chondrocytes exhibited a lower collagen type II expression and a higher collagen type I expression, indicating chondrocyte dedifferentiation.66 The results of this study showed that chondrocyte passage number was an important factor affecting the quality of new cartilagelike tissue formation using chondrocytes.66 This may be one of the explanations why there were no significant differences during MSC cultures under chondrogenic differentiation. As known, the most common method for identifying proteins by proteomic approaches could be the following steps: separation of proteins from a given biological sample by gel electrophoresis, excision of spots from the gel and digestion by trypsin, and extraction from the gel and analysis by mass spectrometry. Protein identification is achieved by mass fingerprint of peptides derived from proteins in databases. But, in a mixture of a high number of peptides, peaks for all components are usually not observed, which leads to poor coverage of the protein sequence. One explanation could be that the presence of one given peptide prevents the response of another. Hydrophilic and hydrophobic interactions of the proteins do not have to rely only on the side chains of the amino acids themselves. By various post-translational modifications, other chains can attach to the proteins, forming hydrophobic lipoproteins or hydrophilic glycoproteins. Therefore, the choice of an adequate matrix plays an important role in peptide desorption as mentioned by Gonnet et al.67 HCCA (R-cyano-4-hydroxycinnamic acid) is a good matrix for peptides with mass ions below Journal of Proteome Research • Vol. 9, No. 10, 2010 5225

research articles 2500 Da, SA (sinapinic acid) is recommended for higher masses (>2500 Da). DHB (2,5-dihydroxybenzoı¨c acid) is recommended for hydrophobic peptides or peptides difficult to be ionized such as glyco- or phospho-peptides. Gonnet et al. have recommended the use of HCCA in concert with SA matrices in order to obtain a good coverage of hydrophilic proteins, while hydrophobic proteins can have their coverage increased by the use of DHB together with SA matrices.67 Thus, the low sequence coverage of some of the proteins presented in this study could be explained by the reasons mentioned above.

Conclusion In summary, we examined the protein expression profiles of rat bone marrow-MSCs differentiated into adipocytes, cardiomyocytes, chondrocytes, and osteocytes. Twenty-three new proteins were identified that were either up- or down-regulated during differentiation experiments, using the MS and SwissProt analysis. Some of the pathways were found to be essential for different progenitor orientations. While some differentiation pathways were held down by particular proteins, some others were disentangled resulting in directed differentiation. Altogether, the findings demonstrate the usefulness of proteomic approaches for understanding various biological processes of the MSCs, including differentiation of the committed progenitors. It seems likely that rat BM-MSCs differentiate through common pathways into adipocytes, chondrocytes, osteocytes, and cardiomyocytes, but further studies are required to clarify the unspecified proteins and their unknown roles.

Acknowledgment. The support of the Turkish ¨ BA (to Y.M.E.), TU ¨ BITAK-BAYG (to Academy of Sciences, TU B. C ¸ .), and Ankara University Biotechnology Institute (Ankara, Turkey) is acknowledged. The authors acknowledge Dr. M. S. Halloran for helpful discussions, and Drs. D. O. Demiralp, and I. Bos¸gelmez for technical support. Supporting Information Available: Spots of interest indicated on a representative 2-DE gels of BM-MSCs (1) adipogenically differentiated at day 7, (2) cardiomyogenically differentiated at day 21, (3) chondrogenically differentiated at day 7, and (4) osteogenically differentiated at day 7. This material is available free of charge via the Internet at http:// pubs.acs.org. References (1) Jiang, Y. H.; Jahagirdar, B. N.; Reinhardt, R. L.; Schwartz, R. E.; Keene, C. D.; Ortiz-Gonzalez, X. R.; Reyes, M.; Lenvik, T.; Lund, T.; Blackstad, M.; Du, J. B.; Aldrich, S.; Lisberg, A.; Low, W. C.; Largaespada, D. A.; Verfaillie, C. M. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002, 418, 41–49. (2) Kolf, C. M.; Cho, E.; Tuan, R. S. Mesenchymal stromal cells. Biology of adult mesenchymal stem cells: regulation of niche, self-renewal and differentiation. Arthritis. Res. Ther. 2007, 9 (1), 204–213. (3) Atmani, H.; Chappard, D.; Basle, M. F. Proliferation and differentiation of osteoblasts and adipocytes in rat bone marrow stromal cell cultures: effects of dexamethasone and calcitriol. J. Cell. Biochem. 2003, 89 (2), 364–372. (4) Pelttari, K.; Steck, E.; Richter, W. The use of mesenchymal stem cells for chondrogenesis. Injury 2008, 39 (Suppl 1), S58–65. (5) Zhang, F. B.; Li, L.; Fang, B.; Zhu, D. L.; Yang, H. T.; Gao, P. J. Passage-restricted differentiation potential of mesenchymal stem cells into cardiomyocyte-like cells. Biochem. Biophys. Res. Commun. 2005, 336 (3), 784–792. (6) Khoo, M. L.; Shen, B.; Tao, H.; Ma, D. D. Long-term serial passage and neuronal differentiation capability of human bone marrow mesenchymal stem cells. Stem Cells Dev. 2008, 17 (5), 883–896. (7) Koc¸, A.; Emin, N.; Elcin, A. E.; Elcin, Y. M. In vitro osteogenic differentiation of rat mesenchymal stem cells in a microgravity bioreactor. J. Bioact. Compat. Polym. 2008, 23 (3), 244–261.

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