Proteomic Profiling of Bone Marrow Mesenchymal Stem Cells upon Transforming Growth Factor β1 Stimulation
2004; Elsevier BV; Volume: 279; Issue: 42 Linguagem: Inglês
10.1074/jbc.m407368200
ISSN1083-351X
AutoresDaojing Wang, Jennifer S. Park, Julia Chu, Ari Krakowski, Kunxin Luo, David J. Chen, Song Li,
Tópico(s)TGF-β signaling in diseases
ResumoBone marrow mesenchymal stem cells (MSCs) can differentiate into different types of cells and have tremendous potential for cell therapy and tissue engineering. Transforming growth factor β1 (TGF-β) plays an important role in cell differentiation and vascular remodeling. We showed that TGF-β induced cell morphology change and an increase in actin fibers in MSCs. To determine the global effects of TGF-β on MSCs, we employed a proteomic strategy to analyze the effect of TGF-β on the human MSC proteome. By using two-dimensional gel electrophoresis and electrospray ionization coupled to quadrupole/time-of-flight tandem mass spectrometers, we have generated a proteome reference map of MSCs, and we identified ∼30 proteins with an increase or decrease in expression or phosphorylation in response to TGF-β. The proteins regulated by TGF-β included cytoskeletal proteins, matrix synthesis proteins, membrane proteins, metabolic enzymes, etc. TGF-β increased the expression of smooth muscle α-actin and decreased the expression of gelsolin. Overexpression of gelsolin inhibited TGF-β-induced assembly of smooth muscle α-actin; on the other hand, knocking down gelsolin expression enhanced the assembly of α-actin and actin filaments without significantly affecting α-actin expression. These results suggest that TGF-β coordinates the increase of α-actin and the decrease of gelsolin to promote MSC differentiation. This study demonstrates that proteomic tools are valuable in studying stem cell differentiation and elucidating the underlying molecular mechanisms. Bone marrow mesenchymal stem cells (MSCs) can differentiate into different types of cells and have tremendous potential for cell therapy and tissue engineering. Transforming growth factor β1 (TGF-β) plays an important role in cell differentiation and vascular remodeling. We showed that TGF-β induced cell morphology change and an increase in actin fibers in MSCs. To determine the global effects of TGF-β on MSCs, we employed a proteomic strategy to analyze the effect of TGF-β on the human MSC proteome. By using two-dimensional gel electrophoresis and electrospray ionization coupled to quadrupole/time-of-flight tandem mass spectrometers, we have generated a proteome reference map of MSCs, and we identified ∼30 proteins with an increase or decrease in expression or phosphorylation in response to TGF-β. The proteins regulated by TGF-β included cytoskeletal proteins, matrix synthesis proteins, membrane proteins, metabolic enzymes, etc. TGF-β increased the expression of smooth muscle α-actin and decreased the expression of gelsolin. Overexpression of gelsolin inhibited TGF-β-induced assembly of smooth muscle α-actin; on the other hand, knocking down gelsolin expression enhanced the assembly of α-actin and actin filaments without significantly affecting α-actin expression. These results suggest that TGF-β coordinates the increase of α-actin and the decrease of gelsolin to promote MSC differentiation. This study demonstrates that proteomic tools are valuable in studying stem cell differentiation and elucidating the underlying molecular mechanisms. Bone marrow is one of the most abundant sources for adult stem cells. Bone marrow mesenchymal stem cells (MSCs) 1The abbreviations used are: MSC, mesenchymal stem cell; TGF-β, transforming growth factor β1; SMCs, smooth muscle cells; SM, smooth muscle; SRF, serum-response factor; LC-MS/MS, liquid chromatography-tandem mass spectrometry; HSP, heat shock protein; FITC, fluorescein isothiocyanate; MS, mass spectrometry; IEF, isoelectric focusing; DTT, dithiothreitol; qPCR, quantitative PCR; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; PBS, phosphate-buffered saline; Q-TOF, quadrupole/time-of-flight; siRNA, small interfering RNA; ddH2O, double distilled H2O.1The abbreviations used are: MSC, mesenchymal stem cell; TGF-β, transforming growth factor β1; SMCs, smooth muscle cells; SM, smooth muscle; SRF, serum-response factor; LC-MS/MS, liquid chromatography-tandem mass spectrometry; HSP, heat shock protein; FITC, fluorescein isothiocyanate; MS, mass spectrometry; IEF, isoelectric focusing; DTT, dithiothreitol; qPCR, quantitative PCR; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; PBS, phosphate-buffered saline; Q-TOF, quadrupole/time-of-flight; siRNA, small interfering RNA; ddH2O, double distilled H2O. are nonhematopoietic and pluripotent stromal cells derived from bone marrow. MSCs can be expanded in culture and differentiate into a variety of cell types such as osteoblasts, chondrocytes, adipocytes, skeletal muscle cells, and smooth muscle cells (SMCs) in response to different microenvironmental cues (1Caplan A.I. Bruder S.P. Trends Mol. Med. 2001; 7: 259-264Abstract Full Text Full Text PDF PubMed Scopus (949) Google Scholar, 2Wakitani S. Saito T. Caplan A.I. Muscle Nerve. 1995; 18: 1417-1426Crossref PubMed Scopus (990) Google Scholar, 3Ferrari G. Cusella-De Angelis G. Coletta M. Paolucci E. Stornaiuolo A. Cossu G. Mavilio F. Science. 1998; 279: 1528-1530Crossref PubMed Scopus (2444) Google Scholar, 4Jiang Y. 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. Aldrich S. Lisberg A. Low W.C. Largaespada D.A. Verfaillie C.M. Nature. 2002; 418: 41-49Crossref PubMed Scopus (5142) Google Scholar, 5Galmiche M.C. Koteliansky V.E. Briere J. Herve P. Charbord P. 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Biol. 2002; 6: 46-50Crossref PubMed Scopus (217) Google Scholar). Proteins separated by two-dimensional electrophoresis can be digested and analyzed by MS, e.g. using matrix-assisted laser desorption ionization (MALDI) or electrospray ionization (ESI) coupled to quadrupole/time-of-flight (Q-TOF) mass analyzers or triple-quadrupole tandem mass spectrometers (MS/MS). MS/MS spectra can be used to determine the peptide sequence with high specificity (18Patterson S.D. Aebersold R.H. Nat. Genet. 2003; 33: 311-323Crossref PubMed Scopus (558) Google Scholar, 22MacCoss M.J. Yates III, J.R. Curr. Opin. Clin. Nutr. Metab. Care. 2001; 4: 369-375Crossref PubMed Scopus (33) Google Scholar). In this study, we used ESI-MS/MS to identify proteins in two-dimensional gels. A preliminary two-dimensional reference map of MSCs was generated, and about 30 TGF-β-regulated proteins with changes at the expression level and/or post-translational modifications were identified. We showed that TGF-β coordinated the increase of SM α-actin and the decrease of gelsolin to promote the assembly of α-actin and actin filaments in MSCs. These results from proteomic profiling will not only provide insight into the global responses of MSCs to TGF-β stimulation but will also lead to in-depth studies on the mechanisms of proteomic changes in MSCs. Cell Culture—Human bone marrow MSCs were obtained from Cambrex Corp. (Walkersville, MD). These MSCs had been well characterized by their surface markers and differentiation potential. They are positive for CD105, CD166, CD29, and CD44 but negative for CD34, CD14, and CD45. MSCs were cultured in MSCGM medium with prescreened fetal bovine serum (Cambrex Corp.) to allow for cell proliferation without differentiation. The cells were maintained in humidified incubators at 37 °C with 5% CO2. Cell culture products and other consumable laboratory supplies were purchased from Fisher and VWR International (Brisbane, CA). MSCs up to passage 10 were used in our experiments. Flow Cytometry—To confirm that MSCs maintain their phenotype after expansion in culture, the cells were subjected to flow cytometry analysis. The cells were detached by trypsin treatment, followed by centrifugation and washing with PBS. After resuspension of the cells, the nonspecific binding sites were blocked by incubation with 1% bovine serum albumin (Sigma) for 30 min. For primary antibodies conjugated with FITC (CD14, CD34, CD45, CD105, and CD166), the samples were incubated with the primary antibody for 30 min, and the expression level of each surface marker was quantified by using a Beckman Coulter EPICS XL flow cytometer. For primary antibodies without FITC conjugation (CD29 and CD44), the samples were incubated with an antibody against each of the surface markers for 30 min and stained with a FITC-conjugated secondary antibody (Molecular Probes, Eugene, OR) for 30 min, followed by flow cytometry analysis. As negative controls, cells were incubated only with the FITC-conjugated secondary antibody. The antibodies against the surface markers CD14 and CD45 were from Santa Cruz Biotechnologies (Santa Cruz, CA). CD34 antibody was from BD Biosciences. CD29 and CD105 antibodies were from Chemicon (Temecula, CA). CD166 antibody was from Serotec (Raleigh, NC). CD44 antibody was from BIOSOURCE (Camarillo, CA). Chemicals and TGF-β Treatment—Chemicals were purchased from Sigma unless otherwise specified. TGF-β1 (Sigma) at 10 ng/ml was used to treat MSCs. Our pilot experiments showed that TGF-β1 at 5 and 20 ng/ml induced similar levels of SM α-actin and collagen I expression in MSCs. For long term culture, TGF-β1 was supplemented when cultured medium was changed (every 2–3 days). Cell Staining and Microscopy—The phase contrast images of MSC morphology were collected by using a Nikon inverted microscope (TE300) with 10× objective and a Hamamatsu Orca100 cooled digital CCD camera. The images were transferred directly from a frame grabber to the computer storage using C-Imaging System software (Compix Inc., Cranberry Township, PA). Immunostaining and confocal microscopy were used to determine the subcellular distribution and organization of the proteins. MSCs were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min, followed by permeabilization with 0.5% Triton X-100 in PBS for 10 min. For immunostaining, the specimens were incubated with the primary antibody against gelsolin (from BD Biosciences), α-actin, or FLAG tag (Sigma) for 2 h and with FITC- or rhodamine-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA) for 1 h. To stain F-actin filaments, the specimen was incubated with rhodamine-phalloidin for 30 min, followed by confocal microscopy. The images of the specimen were collected as Z series sections with a Leica TCL SL confocal microscopy system equipped with argon and He/Ne laser sources, a scanner, and a Leica DM IRB microscope. Multiple sections (0.3 μm thick for each section) were projected onto one plane for presentation. Immunoblotting Analysis of Proteins—To prepare cell lysates for SDS-PAGE, the cells were lysed in a lysis buffer containing 25 mm Tris, pH 7.4, 0.5 m NaCl, 1% Triton X-100, 0.1% SDS, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 1 mm Na3VO4. The lysates were centrifuged at 12,000 rpm by using a microcentrifuge, and the protein concentration of the supernatants was measured by using a DC protein assay (Bio-Rad). The proteins were run in SDS-PAGE and transferred to a nitrocellulose membrane, which was blocked with 3% nonfat milk and incubated with the primary antibody in TTBS buffer (25 mm Tris-HCl, pH 7.4, 60 mm NaCl, and 0.05% Tween 20) containing 0.1% bovine serum albumin. The bound primary antibodies were detected by using a goat anti-mouse or a goat anti-rabbit IgG-horseradish peroxidase conjugate (Santa Cruz Biotechnologies, Santa Cruz, CA) and the ECL detection system (Amersham Biosciences). The immunoblotting results were scanned with a Hewlett Packard high resolution scanner, and the intensity of protein bands were quantified by using NIH Image software. The monoclonal antibody against gelsolin was from BD Biosciences. The antibodies against actin (including all isoforms) and tubulin were from Santa Cruz Biotechnologies. The antibodies against α-actin (monoclonal) and FLAG tag (polyclonal) were from Sigma. The antibody against HSP27 was from Stressgen Biotechnologies Corp. (Victoria, British Columbia, Canada). Cell Lysis and Two-dimensional Gel Electrophoresis—Cells were washed three times using ice-cold PBS buffer and centrifuged down at 3000 rpm for 5 min. Residual PBS buffer on top was removed by careful pipetting. Cells were then disrupted with lysis buffer, which is a mixture of 7 m urea, 2 m thiourea, 4% CHAPS, 40 mm Tris (Tris base), and 20 mm DTT. Normally 1 ml of lysis buffer was used for 1–2 × 107 cells. Upon addition of lysis buffer, cells were immediately pipetted up and down several times to mix well. Samples were allowed to stand at room temperature for about 1 h and vortexed occasionally. They were transferred to Beckman thick wall tubes (362305) and centrifuged at 66,000 rpm (100,000 × g) in a Beckman TLA100.4 rotor for 30 min at 20 °C. Supernatant were aliquoted into siliconized tubes (PGC Scientifics, Frederick, MD) and stored at –80 °C. Modified Bradford assay (Bio-Rad) was used to quantify the total protein amount in the cell lysates. The first-dimension IEF was performed by using an Ettan IPGphor unit (Amersham Biosciences) with a power supply EPS 3501XL. Precast 18-cm, pH 3–10, NL IPG strips were obtained from Amersham Biosciences. 100 μg of lysate mixtures in triplicate were supplemented with rehydration solution (7 m urea, 2 m thiourea, 2% CHAPS, trace of bromphenol blue, 20 mm DTT, and 0.5% corresponding IPG buffer) to a final volume of 350 μl. IPG strips were then rehydrated with the sample mixture in a strip holder for 24 h. IEF was carried out in three steps under step-n-hold mode as follows: (i) 500 V, 1.0 h; (ii) 1000 V, 3 h; and (iii) 8,000 V, 8 h. The total voltage-hour applied was 67,000. The second-dimension SDS-PAGE was carried out in an Ettan DALTsix system (Amersham Biosciences). IPG strips were equilibrated in two consecutive steps: (i) 30 min in 10 mg/ml of DTT; and (ii) 30 min in 25 mg/ml iodoacetamide (Sigma), both dissolved in SDS equilibration buffer (50 mm Tris base, 6 m urea, 30% glycerol (v/v), 2% SDS (w/v), and trace bromphenol blue). 1-mm-thick 10% polyacrylamide gels with a dimension of 27.5 × 21 cm were cast with 30% Duracryl, 0.65% Bis (Genomic Solutions, Ann Arbor, MI), 10% SDS, 10% ammonium persulfate, and 0.375 m Tris buffer at pH 8.8. IPG strips were sealed on the top of gels with 0.5% SeaKem LE-agarose (Cambrex Corp.). SDS-PAGE was performed at a constant voltage of 100 V at 10 °C and stopped once the bromphenol blue front disappeared from the gel. Silver Staining and Image Analysis—Proteins on gels were visualized by using silver staining performed with minor modifications to published procedures (27Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7784) Google Scholar). Briefly, gels were fixed in 50% methanol, 5% acetic acid for at least 2 h followed by a 20-min washing in 50% methanol. Gels were washed twice with ddH2O for 15 min, treated with 0.02% Na2S2O3 for 3 min, and rinsed twice with ddH2O for 1 min before incubation in 0.1% silver nitrate for 30 min. After silver staining the gels were rinsed twice with ddH2O for 2 min and shaken vigorously in developer containing 0.04% formalin (37% formaldehyde in water) and 2% Na2CO3. After 30 s, the developer was discarded, and gels were shaken in fresh developer until the desired intensity was attained (∼3 min). Incubation in 5% acetic acid for 5 min terminated development after which gels were rinsed three times with ddH2O for 2 min prior to imaging. For long term storage, gels were incubated with 1% acetic acid at 4 °C. Stained gels were imaged with an Umax PowerLook 1100 scanner (Umax Technologies, Dallas, TX) with a defined scan resolution of 250 dpi in the transmissive and gray blue mode. Protein expression with and without TGF-β treatment was compared using Z3 3.0 software (Compugen, Tel Aviv, Israel). All gel images were cropped to the same dimensions and auto-contrasted in Photoshop 7.0 prior to image analysis. Multiple gel analysis wizard was applied to compare the two groups of three gels each. Spot detection and matching were achieved initially automatically and were fine-tuned by manual registration. Spurious spots were excluded by manual annotation. To define spots with differential expression, the settings used are as follows: spot contrast of 8, minimum confidence level of 0.95, and minimum spot area (pixels) of 50. Protein spots that were determined to be differentially expressed (n-fold more than 2.0 or less than 0.5) using the automatic analyses were verified manually by local pattern comparison to exclude artifacts. In-gel Tryptic Digestion and Peptide Extraction—To identify differentially expressed proteins, the spots were excised from the gels manually and digested with trypsin. Gel spots were diced into small pieces (1 mm2) and placed into 0.65-ml siliconized tubes. 100 μl (or enough to cover) of 50 mm NH4HCO3, 50% acetonitrile was added, and the tube was vortexed for 10 min. After a brief spin, the supernatant was discarded with gel-loading pipette tips. The washing step was repeated twice. Gel pieces were then brought to complete dryness with a Savant SpeedVac. 5 ng/μl trypsin (Promega, Madison, WI) freshly prepared in 50 mm NH4HCO3 was added to just barely cover the gel pieces. The trypsin solution added was about 3× the volume of dry gel volume estimated, and on average 20 μl was sufficient. The gel pieces were allowed to stand and to swell for 10 min under room temperature. They were then kept on ice for 30 min. Extra trypsin solution was discarded by pipetting to reduce trypsin autolysis. Finally 30 μl of 25 mm NH4HCO3 was gently overlaid on top, and the tubes were incubated at 37 °C overnight (16–20 h). To extract the peptides from the gel pieces, 30 μl of ddH2O was added, and the tube was vortexed for 10 min followed by sonication in a water bath for 5 min. The aqueous portion was transferred to a clean siliconized tube. Peptides were further extracted twice with 30 μl of 50% acetonitrile, 5% formic acid, and supernatants were combined. The total volume was reduced to ∼5 μl by using the SpeedVac. The resultant samples were then subjected to Q-TOF mass spectrometry directly or stored at –20 °C freezer for future analysis. Protein Identification by LC-MS/MS—A hybrid quadrupole/orthogonal time-of-flight mass spectrometer, Q-TOF API US (Waters) interfaced with a capillary liquid chromatography system (Waters), was used to carry out LC-MS/MS analysis. 1–2 μl of samples were injected through an auto-sampler into the LC system at the flow rate of 20 μl/min and pre-concentrated on a 300-μm × 5-mm PepMap C18 precolumn (Dionex, CA). The peptides were then eluted onto a 75-μm × 15-cm PepMap C18 analytical column. The column was equilibrated with solution A (3% acetonitrile, 97% water, 0.1% formic acid), and the peptide separation was achieved with a solution gradient from 3 to 40% of solution B (95% acetonitrile, 5% water, 0.1% formic acid) over 35 min at a flow rate of 250 nl/min. This flow rate through the column was reduced from 8 μl/min from pumps A and B by flow splitting. The LC eluent was directed to the electrospray source with a PicoTip emitter (New Objectives, Woburn, MA). The mass spectrometer was operated in positive ion mode with a source temperature of 100 °C and a cone voltage of 40 V. A voltage of 2 kV was applied to the PicoTip. TOF analyzer was set in the V-mode. The instrument was calibrated with a multipoint calibration by using selected fragment ions from the collision-induced decomposition of Glu-fibrinopeptide B. MS/MS spectra were obtained in a data-dependent acquisition mode in which the three multiple-charged (+2, +3, and +4) peaks with the highest intensity in each MS scan were chosen for collision-induced decomposition. Collision energies were set at 10 and 30 V, respectively, during the MS and MS/MS scans. Mass spectra were processed by using MassLynx 4.0 software, and proteins were identified by using Protein Global Server 1.0/2.0 software. The protein identities were further confirmed by Mascot (www.matrixscience.com) by using the MS/MS peak lists exported from MassLynx. The nonredundant data bases in the molecular mass range of 1,000–500,000 Da and pI values between 3.0 and 10.0 were used at the NCBI website. Modifications considered included carbamidomethylation of cysteine, amino-terminal acetylation, amino-terminal Gln to pyro-Glu, oxidation of methionine, and phosphorylation of serine, threonine, and tyrosine. RNA Isolation and Quantitative PCR (qPCR)—Cells in each well in 6-well plates were lysed with 0.5 ml of RNA Stat 60 (Tel-Test Inc, Friendswood, TX). RNA was extracted using chloroform and phenol extraction steps. Samples were centrifuged inbetween each of these steps for 15 min at 4 °C at 12,000 rpm. Isopropyl alcohol was added to precipitate the RNA, and the samples were centrifuged for 30 min under the same conditions. 75% ethanol was added to wash the RNA pellet and centrifuged at 7500 rpm for 5 min. The pellet was resuspended in 20 μl of diethyl pyrocarbonate-treated water and quantified by using a RiboGreen® RNA quantification assay (Molecular Probes Inc, Eugene, OR). Two-step reverse transcription (RT)-PCR was performed by using the ThermoScript RT-PCR system for first-strand cDNA synthesis (Invitrogen). The cDNA was made from equal amounts of total RNA from each sample, and qPCR was performed by using SYBR green kits and the ABI Prism® 7000 Sequence Detection System (Applied Biosystems, Foster City, CA) (28Li S. Lao J.M. Chen B.P.C. Li Y.S. Zhao Y.H. Chu J. Chen K.D. Tsou T.C. Peck K. Chien S. FASEB J. 2002; 16: U346-U370Google Scholar). Primers for SM α-actin, gelsolin, and 18 S were designed by using the ABI Prism Primer Express™ software version 2.0 (Applied Biosystems). Complete genomic sequences and the mRNA sequence were downloaded from the NCBI website (www.ncbi.nlm.nih.gov/LocusLink) to identify the intron-exon junctions. The primers that span an exon-exon junction were used to ensure the specific amplification of cDNA. The sequences of the designed primers were used to BLAST against nucleotide sequences in the NCBI data base (www.ncbi.nlm.nih.gov/BLAST/) to make sure that the primer sequences were unique. The primers used in this study are listed in Table I.Table IPrimers used in qPCRGene nameForward primer (5′ to 3′)Reverse primer (5′ to 3′)SM α-actinACCCTGCTCACGGAGGCGTCTCAAACATAATTTGAGTCATTTTCTCGelsolinTTGACTTCTGCTAAGCGGTACATCGGCTCAAAGCCTTGCTTCAC18 SCGCAGCTAGGAATAATGGAATAGGCATGGCCTCAGTTCCGAAA Open table in a new tab After each experiment, the melting temperature and the dissociation curve of PCR products were obtained to confirm the product specificity. The amount of RNA for each gene was normalized with the amount of 18 S RNA in the same sample. Transfection of DNA Plasmids and Small Interfering RNA (siRNA)— PCR was used to engineer the FLAG-tagged gelsolin construct by inserting the gelsolin cDNA beginning with the second codon immediately following the FLAG nucleotide sequence (GACTACAAGGATGACGATGACAAG) in the pCMV5b vector. MSCs were seeded in serum-free medium, and the DNA plasmids (2 μg for 10-cm2 culture area) were transfected into MSCs by using the LipofectAMINE PLUS reagent (Invitrogen). After incubation with the mixture of plasmids and LipofectAMINE reagents for 5 h, the cells were cultured for 1 day and treated with TGF-β or kept as control. The expression efficiency of DNA plasmids was about 20%. SiRNA for gelsolin was from Dharmacon Inc. (Lafayette, CO). The FITC-conjugated control siRNA was from Cell Signaling Technology, Inc. (Beverly, MA). The siRNAs (100 nm for 10-cm2 culture area) were transfected into MSCs by using LipofectAMINE 2000. The transfection efficiency of siRNA was more than 90%. Characterization of MSCs—We used MSCs up to passage 10 in our experiments. To confirm that expanded MSCs maintain their phenotype, MSCs were stained for a set of cell surface markers. As shown in Fig. 1, MSCs at passage 10 were positive for CD105, CD166, CD29, and CD44 but were negative for CD14, CD45, and CD34, suggesting that expanded MSCs maintain their phenotype. To further prove that expanded MSCs had pluripotent differentiation potential, MSCs at passage 10 were tested for the differentiation into chondrogenic cells and osteogenic cells by following the instructions supplied by the manufacturer. MSCs cultured as a three-dimensional pellet in chondrogenic media for 2 weeks showed a round shape and synthesized large amounts of glycosaminoglycans, suggesting the differentiation of MSCs into chondrogenic cells (data not shown). MSCs in osteogenic media for 2 weeks showed matrix mineralization by Alizarin Red stain (data not shown), suggesting the differentiation of MSCs into osteogenic cells. TGF-β Induced Morphological Changes, Increased Actin Filaments, and Increased SM α-Actin Expression—Long term treatment of MSCs with TGF-β significantly changed the cell morphology. As shown in Fig. 2, 2 days after TGF-β treatment, MSCs have a more spread out and myoblast-like morphology, and intracellular fibrous structures were visible (indicated by arrows in Fig. 2B). This cell morphology was maintained as the cells grew and reached confluence after 6 days (Fig. 2F). To determine whether the intracellular fibrous structure was actin cytoskeleton, MSCs were stained on actin filaments. Indeed, MSCs trea
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