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Am J Physiol Cell Physiol 292: C342-C352, 2007. First published August 16, 2006; doi:10.1152/ajpcell.00341.2006
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GROWTH, DIFFERENTIATION, AND APOPTOSIS

Stem cell-derived Sca-1+ progenitors differentiate into smooth muscle cells, which is mediated by collagen IV-integrin {alpha}1/beta1/{alpha}v and PDGF receptor pathways

Qingzhong Xiao, Lingfang Zeng, Zhongyi Zhang, Yanhua Hu, and Qingbo Xu

Department of Cardiac and Vascular Sciences, St. George's, University of London, London, United Kingdom

Submitted 20 June 2006 ; accepted in final form 10 August 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Embryonic stem (ES) cells can differentiate into smooth muscle cells (SMCs) that can be used for tissue engineering and repair of damaged organs. However, little is known about the molecular mechanisms of differentiation in these cells. In the present study, we found collagen IV can promote ES cells to differentiate into stem cell antigen-1-positive (Sca-1+) progenitor cells and SMCs. Pretreatment of ES cells with antibodies against collagen IV significantly inhibited SMC marker expression. To further elucidate the effect of collagen IV on the induction and maintenance of SMC differentiation, Sca-1+ progenitor cells were isolated with magnetic beads, placed in collagen-IV-coated flasks, and cultured in differentiation medium with or without platelet-derived growth factor (PDGF)-BB for 6–90 days. Both immunostaining and fluorescence-activated cell sorter analyses revealed that the majority of these cells were positive for SMC-specific markers. Pretreatment of Sca-1+ progenitors with antibodies against integrin {alpha}1, {alpha}v, and beta1, but not beta3, inhibited focal adhesion kinase (FAK) and paxillin phosphorylation and resulted in a marked inhibition of SMC differentiation. Various tyrosine kinase inhibitors, and specific siRNA for phosphatidylinositol 3-kinase (PI 3-kinase) and PDGF receptor-beta significantly inhibited SMC marker expression. Taken together, we demonstrate for the first time that collagen IV plays a crucial role in the early stage of SMC differentiation and that integrin ({alpha}1, beta1, and {alpha}v)-FAK-PI 3-kinase-mitogen-activated protein kinase and PDGF receptor-beta signaling pathways are involved in SMC differentiation.

progenitor cells; extracellular matrix; growth factor receptors; platelet-derived growth factor

ADVANCES IN CELL BIOLOGY, microtechnology, and biomaterial sciences in the past decade have generated new opportunities to create larger and more complex tissues, and eventually complete organs to replace injured or diseased tissues or organs. Vascular cells, especially smooth muscle cells (SMCs), have been isolated from adult arteries and used for engineering vascular tissues. These SMCs divide a finite number of times before undergoing growth arrest in a state known as senescence (27, 43). The limited lifespan of adult SMCs may therefore be the rate-limiting step in constructing autologous human vessels in vitro to replace injured vascular tissue. The finding of a new cell source to obtain large amounts of SMCs is important to develop vascular tissue engineering for the treatment of vascular disease.

Embryonic stem (ES) cells are the pluripotent derivatives of the inner cell mass of blastocysts. They have the capacity for unlimited growth and self-renewal and the ability to differentiate into all types of mature tissue cells, including germ cells. In the last several years, accumulating evidence indicates that ES cells can differentiate into endothelial cells in vitro or in vivo (1, 8, 17, 18, 24, 26, 35, 40, 45, 49–51). Drab et al. (13) demonstrated that retinoic acid and dibutyryl-cAMP enhanced SMC differentiation from mouse ES cells. Yamashita et al. (49) and Sone et al. (40) demonstrated that vascular endothelial growth factor receptor-2-positive (VEGFR2+) progenitor cells from differentiated ES cell cultures can differentiate into both endothelial cells and SMCs. Little is known about the molecular mechanisms of ES cell differentiation into SMCs, and no data exist concerning how a large number of SMCs with high purity can be produced from ES cells in vitro.

The extracellular matrix (ECM) is a complex structural entity surrounding and supporting cells that are found within mammalian tissues. Previous studies demonstrated that ECM is an important factor affecting cell adherence (21), growth (21), migration (21), apoptosis (9), and differentiation (15). ECM is composed of three major classes of biomolecules, including structural proteins (collagen and elastin), specialized proteins (fibrillin, fibronectin, and laminin), and proteoglycans. Collagens are the most abundant proteins found in the animal kingdom and are the major proteins comprising the ECM. There are at least 12 types of collagen. Types I, II, and III are the most abundant and form fibrils of similar structure. Type IV collagen forms a two-dimensional reticulum and is a major component of the basal lamina. Previous studies have shown that collagen type IV has a crucial role in the early stage of differentiation of F9 stem cells (46). Yamashita et al. (49) and Sone et al. (40) demonstrated that VEGFR2+ progenitor cells can differentiate into SMCs when cultured in collagen IV-coated dishes and {alpha}-minimal essential medium (MEM) containing 10% FBS. However, the functional role of collagen type IV in SMC differentiation in ES cells is not clear.

Integrins are a family of noncovalently associated heterodimeric cell-surface receptors composed of {alpha}- and beta-subunits that mediate cell-ECM and cell-cell adhesion. There are 18 {alpha}- and 8 beta-subunits, excluding splice variants, that combine to form more than 24 different integrins (33). Integrins not only support cell attachment but also act in concert with receptors for soluble factors to regulate cell survival, proliferation, apoptosis, and differentiation (11). However, it is not clear how integrin signaling pathways mediate ES cell differentiation into SMCs in vitro or in vivo.

In a previous study, we demonstrated that a population of vascular progenitor cells are present in the adventitia, which can differentiate into SMCs that contribute to atherosclerosis (20). We further indicated that stem cell antigen-1 (Sca-1) can be used as a sorting marker of SMC progenitors to isolate these cells from vascular tissues (20). Sca-1 is a cell surface protein used to identify hematopoietic stem cells and has been localized to the vasculature in some tissues (42). Progenitor cells expressing Sca-1 in a mouse model were shown to differentiate into SMC in vitro and in vivo (20). We hypothesized that Sca-1-positive (Sca-1+) cells derived from ES cells can also serve as SMC progenitors to produce SMCs in vitro. We also hypothesized that collagen type IV has a functional role in SMC differentiation of ES cells and that the collagen type IV-integrin signaling pathways mediate SMC differentiation from Sca-1+ progenitor cells. In the present study, we isolated Sca-1+ cells from mouse ES cells, differentiated the progenitors, and characterized these cells. We demonstrated that a large number of SMCs with high purity can be prepared from ES cells and that SMC differentiation involves the collagen IV-integrin ({alpha}1, beta1, and {alpha}v)-focal adhesion kinase (FAK)/paxillin-phosphatidylinositol 3-kinase (PI 3-kinase)-mitogen/extracellular signal-regulated kinase (MEK)-extracellular signal-regulated kinase (ERK)/c-Jun/NH2-terminal kinase (JNK) signaling pathways.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. Detailed information of all the reagents used in this study, including inhibitors, antibodies, and RGD peptides is shown in supplemental Table 1. (The online version of the paper contains supplemental data.) In brief, the primary antibodies against smooth muscle {alpha}-actin (SMA; 1:5,000), calponin (1:10,000), and smooth muscle myosin heavy chain (SMMHC; 1:2,000) were purchased from Sigma (St. Louis, MO). Phosphopaxillin (Tyr118) was purchased from Cell Signaling Technology (Beverly, MA), and phospho-FAK (Tyr397) was from Upstate Biotechnology (Waltham, MA). P38 mitogen-activated protein kinase (MAPK)-specific inhibitor SB-202190, JNK-specific inhibitor SP-600125, MEK-specific inhibitor PD-98059, and tyrosine kinase inhibitor set II (AG18T9, AG-490, AG-1296, and AG-1478; Genistein) were purchased from Calbiochem (VWR International). RGD peptides G4391 (antagonist of integrin function) and S3771 (negative control for G4391), PI 3-kinase-specific inhibitor LY-294002, and cytochalasin B were from Sigma.

ES cell culture. Mouse ES cells (ES-D3 cell line, CRL-1934; ATCC, Manassas, VA) were maintained as described previously (45). Briefly, ES cells were cultured in DMEM (ATCC), supplemented with 10 ng/ml recombinant human leukemia inhibitory factor (LIF; Chemicon, Temecula, CA), 10% FBS (ATCC), 2 mM L-glutamine (Sigma), 0.1 mM 2-mercaptoethanol (Sigma), 100 U/ml penicillin, and 100 mg/ml streptomycin (GIBCO, Grand Island, NY). Undifferentiated cells were fed every 2 days with fresh media and passaged into new gelatin (Sigma)-coated flasks with a 1:6 to 1:10 ratio every 2–4 days. In some experiments, ES cells were replated in 5 µg/ml of different ECM (fibronectin, collagen I, and collagen IV) and poly-L-lysine-coated dishes or flasks and cultured in differentiation medium [DM; {alpha}-MEM (GIBCO) supplemented with 10% FCS (GIBCO) and 0.05 mM 2-mercaptoethanol (Sigma)] for 2, 4, 6, 10, and 14 days. Cells were fed with fresh DM every day.

Cell sorting and differentiation. ES-D3 cells (1 x 106) were cultured on type IV mouse collagen (Trevigen, Gaithersburg, MD)-coated flasks (surface area: 75 cm2) in basic DM for 3–4 days. As described in our previous studies(20), Sca-1+ cells were sorted from the cell culture by magnetic labeling cell sorting with anti-Sca-1 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, ES cells cultured in collagen IV and DM for 3–4 days were detached with a 0.05% trypsin-EDTA solution (GIBCO) from flasks and incubated with the antibody-conjugated/coated microbeads at 4°C with occasional agitation for 15 min. The bead-bound cells were then selected using a magnetic cell separator (Miltenyi Biotec). Sca-1+ cells were resuspended and cultured in fresh ES cell growth medium. To obtain higher numbers of Sca-1+ cells, these cells were expanded in ES cell culture medium and resorted with anti-Sca-1 microbeads. For SMC differentiation, Sca-1+ cells were plated on collagen IV-coated dishes or flasks, cultured in basic DM or basic DM plus 10 ng/ml platelet-derived growth factor (PDGF)-BB (Sigma) for 6 days (first passage), and then passaged every 3 days. For long-period culture of ES cell-derived SMCs (esSMCs), cells grown to >80% confluence were passaged into new collagen-coated flasks with a 1:2 to 1:3 ratio every 2–3 days and cultured in DM. In some experiments, Sca-1+ progenitor cells were pretreated with RGD peptides or functional blocking antibodies against integrins or cultured in collagen IV-coated dishes and DM in the presence of various specific inhibitors for 6 days.

RT-PCR. Total RNA was extracted from cells, mouse spleen, and mouse aorta using RNeasy kits (Qiagen, Valencia, CA) according to the manufacturer's instructions. RNA was quantified with an ultraviolet spectrophotometer, and cDNA was synthesized from 2.5 µg total RNA for each RT reaction. Reverse transcription was performed using an Improm-II RT kit (Promega, Madison, WI) with RNase inhibitor (Promega) and Random primers (Promega). Simultaneous RT reactions were performed without the addition of RT to control for the transcription of contaminating genomic DNA. Fifty nanograms of cDNA (relative to RNA amount) were used to perform PCR with a PCR kit (Invitrogen, San Diego, CA) following the manufacturer's instructions. Oligonucleotide primer sequences were shown in supplemental Table 2.

Immunofluorescence staining. The procedure used for immunofluorescent staining was similar to that described previously (20). Briefly, esSMCs were labeled with mouse isotype IgG control or mouse monoclonal antibodies to SMA (clone 1A4; Sigma), calponin (clone hCP; Sigma), and SMMHC (clone HSM-V; Sigma) and visualized with rabbit anti-mouse Ig conjugated with FITC (DAKO) or phycoerythrin (DAKO). Cells were counterstained with 4',6-diamidino-2-phenylindole (DAPI; Sigma), mounted in Floromount-G (Cytomation; DAKO, Glostrup, Denmark), and examined with a fluorescence microscope (Axioplan 2 imaging; Zeiss). Human umbilical vein endothelial cells (HUVECs) were used as cell negative controls.

Flow cytometry analysis. The procedure used for flow cytometry was similar to that described previously (20). Briefly, cultured cells were harvested by incubating cell cultures with dissociation buffer (GIBCO). The harvested cells were fixed, permeabilized with 0.1% Triton X-100, and incubated in diluted serum (1:10, the species of serum is the same as the secondary antibody that was used) for 20 min on ice to block any nonspecific antibody binding. The single-cell suspension was separated into aliquots and incubated with either isotype control or stage-specific embryonic antigen-1 (SSEA-1), Sca-1, SMA, calponin, and SMMHC antibodies for 30 min on ice, followed by incubation with rabbit anti-mouse Ig conjugated with FITC (DAKO) or rabbit anti-rat Ig conjugated with FITC (DAKO). Cell suspensions were analyzed with a fluorescence-activated cell sorter analysis (FACS) scan flow cytometer (Becton-Dickinson Immunocytometry Systems, Mountain View, CA). Forward and 90° side scatter were used to identify and gate positive and negative fractions. Data analysis was carried out using CellQuest software (Becton-Dickinson).

Western blot analysis. Protein (40 µg) was separated by SDS-PAGE with 4~20% Tris-glycine gel (Invitrogen, Carlsbad, CA) and transferred to nitrocellulose transfer membranes (Schleicher & Schuell Bioscience). The membranes were blocked with PBS or TBS containing 5% dry milk and 0.1% Tween 20 on a shaker at room temperature for 1 h and probed with appropriate primary antibodies at 4°C overnight, followed by incubation with the secondary antibody at room temperature on a shaker for 1 h. The membrane was developed using ECL-Plus reagent (Amersham Biosciences, Stockholm, Sweden). Blots were stripped and reprobed with {alpha}-tubullin as equal loading control.

Small interfering RNA knockdown experiment. PDGF receptor-beta (PDGFRbeta) ShortCut small interfering RNA (siRNA) Mix (N2023S) was purchased from New England Biolabs (Beverly, MA). The control siRNA (no. 4611) and the siRNA for PI 3-kinase (sense: 5'-gaguguggagaaggagacutt-3'; antisense: 5'-agucuccuucuccacacuctt-3') were purchased or synthesized from Ambion (Huntingdon, Cambridgeshire, UK). For the PDGFRbeta siRNA knockdown experiments, ES cells were cultured in 5 µg/ml of fibronectin-coated dishes for 2–3 days, and the medium was refreshed at 24 and 1 h before transfection. Cells were transfected with 50 nM of control siRNA and PDGFRbeta siRNA Mix at days 3 and 7. Cells were harvested at day 10, and the protein levels of SMA were detected by Western blot analysis. For the PI 3-kinase siRNA knockdown experiments, Sca-1+ cells were cultured on a collagen IV-coated six-well plate for 2–3 days, and 5 µl of 20 µM siRNA/well (100 nM of final concentration) were introduced in the cells with siIMPORTER transfection reagents (Upstate) according to the protocol provided. The control siRNA-transfected cells were included as control. For specific target gene, siRNA transfection was carried out in duplicate. Cells were harvested at day 6, and the protein levels of SMA were detected by Western blot. Approximately 85% of transfection efficiency was achieved in our siRNA knockdown experiment.

Statistical analysis. Data expressed as means ± SE were analyzed with a two-tailed Student's t-test for two groups or one-way ANOVA for different groups. A value of P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Collagen IV can promote ES cell differentiation into SMCs. To test the functional effects of matrix proteins on initial SMC differentiation, ES cells were placed in the dishes or flasks coated with 5 µg/ml of differential ECM (collagen IV, collagen I, and fibronectin) and poly-L-lysine and cultured in DM for 2, 4, 6, 10, and 14 days. ES cells cultured in collagen-I-, collagen-IV-, and fibronectin-coated dishes were adhesive and grew well but died in poly-L-lysine-coated dishes in the first 4 days of culture. As shown in Fig. 1, A–C, although all three ECMs were able to induce ES cells to differentiate into SMC, collagen IV promoted SMC differentiation in the early stage of SMC differentiation (from day 6, the protein levels of SMA were gradually increased). Additionally, FACS analysis of the percentage of SMA-positive cells in the differentiated ES cells demonstrated that collagen IV significantly promoted SMC differentiation (P < 0.05; Fig. 1D). Finally, the protein levels of progenitor cell marker (Sca-1) were upregulated from day 2 and then downregulated from day 6 of differentiation, indicating that a Sca-1+ progenitor population is generated in the early stage of collagen IV-mediated SMC differentiation. On the other hand, the protein levels of Sca-1 appeared later and much weaker in collagen I- and fibronectin-mediated SMC differentiation (Fig. 1, B and C).


Figure 1
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  		Fig. 1. Collagen IV plays a crucial role in smooth muscle cell (SMC) differentiation. A–E: embryonic stem (ES) cells were plated in dishes coated with 5 µg/ml of collagen IV, collagen I, fibronectin, or poly-L-lysine and cultured for a variety of times. Cells were harvested, and SMA, stem cell antigen-1 (Sca-1), and collagen IV proteins were detected by Western blot analysis (A–C and E). As controls, mouse vascular SMC (mSMC), spleen, and aorta tissues were used. Data were representative of 2 independent experiments. {alpha}-Tubulin serves as loading controls. In some experiments, cells were harvested, and the percentage of SMA-positive cells was analyzed by FACS (D). Data shown in D are means ± SE (n = 3 experiments). *P < 0.05, collagen (Col) IV vs. collagen I or fibronectin (Fibro). F: ES cells were cultured in 5 µg/ml of fibronectin-coated dishes overnight, and 5 µg/ml of control (Ctrl) IgG or neutralizing antibody (Abs) against collagen IV were added at 2nd day and cultured for 9 days. Cells were harvested at day 10, and SMA proteins were detected by Western blot. F, top: representative data; F, bottom: means ± SE of densitometric analysis (n = 3), *P < 0.05. MW, mol mass marker.

 
Because collagen I and fibronectin can also stimulate ES cells to differentiate into SMCs (Fig. 1, B and C), we hypothesized that cells in these conditions may produce collagen IV that results in cell differentiation. To verify this hypothesis, we examined the expression of collagen IV with Western blot analysis. Data shown in Fig. 1E indicates that collagen IV proteins were detected as early as day 4 in both collagen I- and fibronectin-coated cultures. Interestingly, compared with control IgG, when specific neutralizing antibody against collagen IV was applied to treat cells in day 2 of culture, no significant differences of cell morphology or cell adhesion were observed; however, SMA expression was inhibited significantly by function-neutralizing antibody (P < 0.05; Fig. 1F). Taken together, these results indicate that collagen IV plays a crucial role in SMC differentiation from ES cells.

Isolation and characterization of esSMCs. Previously, we showed that Sca-1+ cells derived from adventitial tissues can differentiate into SMCs (20). Moreover, we found that a Sca-1+ subprogenitor population existed in the early stage of collagen IV-mediated SMC differentiation (Fig. 1A). To test whether Sca-1+ cells from ES cells have a similar ability to differentiate into SMCs, ES cells were plated in collagen IV-coated flasks and cultivated in DM for 3–4 days, and Sca-1+ cells were isolated with microbeads. FACS analysis of ES cells demonstrated that 96.5% cells were SSEA-1 positive, but not SMC marker {alpha}-actin (Fig. 2A). A small fraction (8.6%) of cells were Sca-1+ after 3-day culture in collagen IV-coated flasks, whereas the majority (>94%) of cells were Sca-1+ after isolation with magnetic beads coupled with anti-Sca-1 antibodies (Fig. 2B). At this stage, SMC markers, e.g., SMA, calponin, and smoothelin, were absent in Sca-1+ cells (Fig. 2E). Sca-1+ cells were maintained in ES cell culture medium to be further expanded and used in other experiments.


Figure 2
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  		Fig. 2. Characteristics of ES cell-derived SMCs (esSMCs). Sca-1-positive (Sca-1+) cells were sorted out from ES cell-derived cultures (see MATERIALS AND METHODS) and cultured in ES cell medium to prevent further differentiation. For SMC differentiation, Sca-1+ cells were cultured in differentiation medium (DM) with 10 ng/ml of platelet-derived growth factor (PDGF)-BB for 6 days (esSMC-passage 1) or more (esSMCs, as described in text). ES cells (A), pre- and postsorted Sca-1+ cells (B), and esSMCs (D) were labeled with antibodies and analyzed by fluorescence-activated cell sorter (FACS) analysis. FL1-H, green fluorescence intensity; FL2-H, yellow fluorescence intensity. Data shown in D are means ± SE (n = 3). esSMC (passage 10) were also stained for smooth muscle {alpha}-actin (SMA; red), calponin (red), smooth muscle myosin heavy chain (SMMHC; red), and mouse isotype IgG control (C). Nuclei were counterstained with 4',6-diamidino-2-phenylindole (blue); original magnification x400. SMC specific markers were detected by Western blot analysis with antibodies against SMA, calponin, and smoothelin (E). mRNA levels of SMMHC isoform 1 and 2 (SM1 and SM2, respectively) were detected by RT-PCR (F). As controls, spleen, aorta, and atherosclerotic (AS) vessels from apoE-deficient mice, Sca-1+ cells, and mouse vascular SMC (mSMC) were included in Western blot analysis. Pn, passage no. Control antibodies macrophage-specific marker (Mac-1), CD144, and CD45 were also used. {alpha}-Tubulin and beta-actin serve as loading controls.

 
For SMC differentiation, Sca-1+ cells were cultured in DM with 10 ng/ml of PDGF-BB for 6 days (these cells were defined as esSMC-passage 1) and then were further cultured with a split ratio of 1:2 or 1:3 every 2–3 days in DM without PDGF-BB [labeling the cell as esSMC-passage(n+1)]. The passage numbers reached 10, and the expected total cell numbers of esSMCs reached 1–3 x 109 after 25–30 days of differentiation. Morphologically, Sca-1+ progenitors and esSMCs displayed a monolayer in culture, whereas ES cells showed clusters in an undifferentiated state for >35 passages in our culture conditions. In addition, Sca-1+ cells grew as round cells, and the morphology of esSMCs was similar to that of adult medial SMCs, which displayed a hill-and-valley pattern (supplemental Fig. 1). Immunostaining indicated that ES cells were positive for ES specific marker SSEA-1 after 20 passages, but not Sca-1 (data not shown). When isolated Sca-1+ cells were cultured in DM with PDGF-BB for 6 days, the majority of esSMCs were positive for SMA, calponin, and SMMHC (Fig. 2C), but HUVECs were negative for SMA, calponin, and SMMHC (data not shown).

To evaluate the purity of differentiated SMCs derived from ES cells, we quantitatively analyzed the expression of SMC specific markers with flow cytometry. As shown in Fig. 2D, approximately 55~66% of cells expressed SMC markers after treatment with DM containing 10 ng/ml PDGF-BB for 6 days. As expected, the percentage of positive cells for SMC markers increased with longer periods of culture, and >95% of the differentiated SMCs at passage 10 were SMA, calponin, and SMMHC positive (Fig. 2D). To further validate the specificity of antibodies against SMC-specific markers, including SMA, calponin, and smoothelin, high-resolution Western blot analysis with a complete set of negative and positive controls for SMC-specific markers was conducted with these antibodies. As shown in Fig. 2E, high levels of SMC-specific proteins were expressed in differentiated SMCs (esSMC), and in even higher passage cells (esSMC-p30), but were not expressed in Sca-1+ cells. To further investigate the purity of esSMCs, the cells were examined for the expression of endothelial cell-specific marker (CD144), leukocytes common antigen (CD45), and macrophage-specific marker (Mac-1) with Western blot analysis. As shown in Fig. 2E, all three markers were not expressed in esSMCs.

To further characterize esSMCs, SM1 and SM2, two markers of mature contractile SMCs, were detected by RT-PCR with the specific primers, as described in the previous report (23). As shown in Fig. 2F, both were detected in esSMCs, although SM2 expression was weaker than SM1. This result further supports the fact that esSMCs with a high purity and maturation can be obtained from Sca-1+ progenitor cells using these experimental conditions.

Role of integrins in Sca-1+ progenitor cell differentiation into SMCs. To study the molecular mechanisms involved in SMC differentiation, ES cell-derived Sca-1+ progenitor cells were cultured in collagen IV-coated dishes in DM with or without PDGF-BB. Protein levels of SMC-specific markers gradually increased in a time-dependent pattern (Fig. 3A). Because collagen can specifically bind to integrins, we examined their expression during SMC differentiation. As shown in Fig. 3B, both ES cell-derived Sca-1+ progenitor cells and esSMCs expressed high levels of some integrins, such as integrin {alpha}1, {alpha}v, beta1, and beta3, but not {alpha}2 and beta2 (data not shown). To further investigate the functional involvement of these four types of integrins, Sca-1+ progenitor cells were plated in the collagen IV-coated dishes and treated with RGD peptide G4391, RGD negative control peptide S3771, control IgG, and blocking antibodies against integrin {alpha}1, {alpha}v, beta1, and beta3 alone from the 2nd day of differentiation. Data of Western blot analyses shown in Fig. 3C indicate that both RGD peptide G4391 and antibodies against integrin {alpha}1, {alpha}v, and beta1, but not beta3, markedly inhibited SMC marker expression (P < 0.01), implicating the essential role of these integrin members in progenitor cell differentiation into SMCs.


Figure 3
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  		Fig. 3. Role of integrin ({alpha}1, beta1, and {alpha}v) in SMC differentiation. Sca-1+ progenitor cells were cultured in collagen IV-coated dishes with or without 10 ng/ml of PDGF-BB. SMC-specific marker (A) and integrin (B) proteins were detected by Western blot analysis. C: Sca-1+ progenitor cells were plated in the collagen IV-coated dishes and treated with RGD peptide (G4391, 0.5 mM), RGD peptide negative control (S3771, 0.5 mM), 50 µg/ml of antibodies against integrins, or normal IgG (IgG control) from day 2 to 6. Cells were harvested, and the proteins levels as indicated were detected by Western blot analysis. C, bottom: means ± SE. P < 0.05 vs. IgG controls (*) and vs. vehicle controls (#).

 
Previous studies showed that activation of integrins promotes the formation of membrane adhesion complexes, known as focal adhesions, including cytoskeletal proteins and protein tyrosine kinases, e.g., FAK and paxillin (30). To clarify whether integrin-mediated signaling is required for SMC differentiation, we examined the activation of FAK and paxillin. As shown in Fig. 4A, FAK and paxillin were phosphorylated during SMC differentiation. Treatment of Sca-1+ progenitor cells with RGD peptide G4391 and antibodies against integrin {alpha}1, {alpha}v, and beta1, but not beta3, markedly or completely inhibited phosphorylation of FAK and paxillin (Fig. 4B). To further clarify the functional role of the integrin/FAK pathway, Sca-1+ progenitor cells were treated with cytochalasin B to disrupt actin filaments and integrin signaling or incubated with FAK inhibitor PP2 and genestein. Cytochalasin B completely blocked SMC differentiation (Fig. 4C), whereas PP2 and genestein significantly inhibited the phosphorylation of FAK (Fig. 4B), which led to blocking of SMC differentiation (Fig. 4C).


Figure 4
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  		Fig. 4. Focal adhesion kinase (FAK)/paxillin and PDGF receptor-beta (PDGFRbeta) activation in SMC differentiation. A: Sca-1+ progenitor cells were replated in collagen IV-coated dishes and cultured in DM for 2, 4, and 6 days. Phosphorylated FAK (p-FAK) and paxillin (p-paxillin) were detected by Western blot analysis. Total FAK and Pan paxillin serve as loading controls. B: Sca-1+ progenitor cells were treated with vehicle (normal IgG and 0.1% DMSO), FAK inhibitor (PP2, 10 µM), RGD peptide (G4391), and antibodies against integrin {alpha}1, beta1, {alpha}v, and beta3. Phosphorylated FAK and paxillin were detected by Western blot (left) and semiquantified for protein levels (right). *P < 0.01 (n = 3). C: Sca-1+ progenitor cells were cultured in the presence of DMSO, cytochalasin B, genistein, PP2, AG-1296, AG-490, AG-1478, or suramin for 6 days. SMC marker proteins were detected by Western blot analysis (top) and semiquantified for protein levels (bottom). *P < 0.01 (n = 3). D: ES cells were cultured in DM for 2–3 days, and cells were transfected with 50 nM of control small interfering RNA (siRNA) or PDGFRbeta siRNA at days 3 and 7. Cells were harvested at day 10, and the protein levels of PDGFRbeta and SMA were detected by Western blot analysis (left). Graphic data are means ± SE of SMA/{alpha}-tubulin ratio (right). *P < 0.05 (n = 3).

 
Involvement of tyrosine kinases. Besides integrin signaling, growth factor receptors, such as PDGFRbeta and epithelial growth factor (EGF) receptor, might also be involved in SMC differentiation from Sca-1+ progenitor cells. When Sca-1+ progenitor cells were treated with suramin [suramin inhibits cell-surface binding of EGF, PDGF, transforming growth factor (TGF)-beta, and VEGF receptors as well as serotonin], PDGFR-specific inhibitor AG-1296, or EGF receptor-specific inhibitors AG-490 and AG-1478, expression of proteins of SMC-specific markers was significantly or completely blocked (Fig. 4C). These results suggested that both growth factor receptor and nongrowth factor receptor tyrosine kinase-mediated signaling is involved in SMC differentiation.

Previously, reports demonstrated that soluble mitogenic growth factor PDGF-BB plays an important role in SMC differentiation in vivo and in vitro. However, we did not observe a significant effect of addition of 10 ng/ml of PDGF-BB on SMC differentiation in our experimental system with 10% FBS (Fig. 3A). Because the presence of PDGF-BB in serum supplemented to the medium cannot be ruled out, we investigated whether the PDGFR is involved in SMC differentiation using a PDGFRbeta-specific siRNA technique. Transfected cells with PDGFRbeta-specific siRNA displayed a significant inhibition of SMA expression in SMC differentiation (P < 0.05; Fig. 4D).

Because PI 3-kinase is downstream of tyrosine kinase receptors and FAK, we further investigated the involvement of PI 3-kinase in SMC differentiation. The protein level of PI 3-kinase was found to increase during SMC differentiation and reached a peak at 4 days (data not shown), but GTPase-binding protein Ras did not change significantly. Treatment of Sca-1+ progenitor cells with PI 3-kinase-specific inhibitor LY-294002 resulted in a decrease in SMC-specific marker protein expression in a dose-dependent pattern (Fig. 5A). In addition, treatment of cells with LY-294002 completely inhibited the phosphorylation of ERK1/2, JNK, and c-Jun, but much less for p38MAPK (Fig. 5B), indicating ERK1/2 and JNK are downstream of PI 3-kinase in current SMC differentiation. Furthermore, we have performed siRNA knockdown experiments with specific siRNA for PI 3-kinase. Sca-1+ progenitors treated with specific siRNA for PI 3-kinase, not control siRNA, revealed significant inhibition of SMC differentiation (P < 0.05), which is consistent with the result from PI 3-kinase-specific inhibitor LY-294002 (Fig. 5C). Similarly, inhibition of ERK1/2 or JNK, but not pP38MAPK activation with specific inhibitors resulted in decreased expression of SMC-specific markers with a dose-dependent pattern (Fig. 5D). The effect of these specific inhibitors applied in the present study with indicated concentration on Sca-1+ progenitor cell viability, adhesion, and morphology is very small; no significant differences were observed between control and various inhibitor treatments (data not shown).


Figure 5
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  		Fig. 5. Phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein kinase (MAPK) are involved in SMC differentiation. A: Sca-1+ progenitor cells were treated with LY-294002 for 6 days. SMC-specific markers were detected by Western blot analyses. Left: representative images; right: means ± SE; n = 3. *P < 0.05. B: cells were treated as indicated in A. Left: representative images; right: means ± SE; n = 3. ERK, extracellular signal-regulated kinase; JNK, c-Jun/NH2-terminal kinase. *P < 0.05. C: Sca-1+ cells were cultured in 5 µg/ml of collagen-IV-coated dishes for 3 days. Cells were transfected with 100 nM of control siRNA or PI 3-kinase-siRNA. Cells were harvested at day 6, and the protein levels of PI 3-kinase, SMA, and calponin were detected by Western blot analysis. Left: representative images; right: means ± SE; n = 3. *P < 0.05. D: Sca-1+ progenitor cells were treated with 0.1% DMSO (vehicle control), SB-202190, SP-600125, or PD-98059 for 6 days. SMC-specific markers were detected by Western blot analysis. Left: representative images; right: means ± SE; n = 3. *P < 0.01.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we have developed a method for producing a large number of SMCs from stem cells and demonstrated for the first time that Sca-1+ progenitor cells derived from ES cells can differentiate into SMCs with high purity (>95%). We demonstrated that collagen IV-integrin- and PDGFRbeta-mediated signaling pathways are important for Sca-1+ progenitor cell differentiation into SMCs. Our results provide the possibility to generate SMCs from stem cells that can be used as a source of cells for vascular engineering and repair of injured vessels.

It is recognized that a highly purified cell population is a key issue for successful tissue engineering. In the present study, we found that continued culture of differentiated esSMCs for >30 days is needed to obtain a large number of SMCs with high purity (>95%). Additionally, these esSMCs only express high levels of SMC markers, but not other cell-specific markers, such as endothelial cell-specific marker (CD144), leukocyte common antigen (CD45), and Mac-1, even in higher passages (esSMC-p30). Importantly, esSMCs still display high proliferative properties even after 35 passages of culture. These results indicate that the method of SMC differentiation we developed in this study is effective for producing functional SMCs from stem cells.

Atherosclerosis is an inflammatory disease in which risk factors such as hyperlipidemia, hypertension, diabetes, smoking, and infections can directly or indirectly stimulate the arterial endothelium, resulting in its dysfunction, damage, or both. Once the integrity of the endothelium is interrupted, lipid penetration and mononuclear cell adhesion might be initiated. Traditionally, it was believed that the damaged endothelial cells would be replaced by neighboring endothelial replication and SMCs from the media would migrate in the intima to constitute the lesions. This concept is challenged, however, by the discovery that stem/progenitor cells in the circulation and adventitia contribute to endothelial repair and SMC accumulation. Stem cells/progenitor cells from blood and the adventitia migrate into the intima, where they proliferate and differentiate into neo-SMCs to form neointima (48). Although PDGF-BB is expressed at low levels in arteries from healthy adults, its expression is increased in the local atherosclerotic lesion. Many studies of balloon catheter-injured arterial tissue, naturally occurring atherosclerosis, coronary arteries after percutaneous transluminal coronary angioplasty, and experimentally induced atherosclerosis revealed increased expression of PDGF-BB and PDGF receptors in these lesions. These observations suggest that PDGF-BB, produced by activated macrophages, SMCs, or endothelial cells or released from platelets in thrombi, is important for the formation of the lesion. PDGF-BB will be released from platelets, which aggregate to the damaged endothelium. The role of PDGF-BB in atherosclerotic lesions may be to stimulate SMCs to migrate from the media of the vessel to the intima layer and to proliferate and produce matrix molecules at this site, as well as to induce stem/progenitor cells migrated from blood or adventitia to differentiate into mature SMCs and then result in neointima formation (16).

Bradfute and colleagues (4) observed that Sca-1 plays a key role in hematopoietic progenitor/stem cell lineage fate and c-kit expression. Overexpression of Sca-1 affects stem/progenitor cell activity (4). In our previous study, we demonstrated that isolated Sca-1+ progenitor cells from adventitial cells were able to differentiate into mature SMCs in response to PDGF-BB stimulation in vitro (20). Our study also used Sca-1+ selected cells from smooth muscle-LacZ mice so that only when they differentiated to SMC they would be identifiable by X-gal staining to demonstrate that Sca-1+ cells can differentiate into SMCs that contribute to neointima formation in vivo. Moreover, other groups demonstrated that Sca-1+ progenitor cells can differentiate into endothelial cells (34), hematopoietic cells (19), and skeletal muscle myotubes (32). In our preliminary study, we observed that Sca-1-negative cells were senescent after five to six passages; however, Sca-1+ cells retained unlimited growth properties, even after 30 passages, which indicates that Sca-1+ progenitor cells have an important role in SMC differentiation.

Many reports in the literature demonstrate that soluble mitogenic growth factors are necessary for SMC differentiation in vivo and in vitro, such as PDGF-BB (20, 37, 40, 49) and TGF-beta (7, 22, 38). In the present study, we found that the effect of exogenous PDGF-BB added to the culture medium on SMC differentiation is weak and that PDGF receptor-mediated signaling is essential for SMC differentiation. Treatment of Sca-1+ progenitor cells with either growth factor receptor antagonist or growth factor receptor tyrosine kinase specific inhibitors, including suramin, AG-1296, AG-490, and AG-1478, significantly or completely blocked SMC differentiation. How can these phenomena be explained? One possibility is that a certain amount of growth factors, e.g., PDGF-BB and EGF, exist in the serum supplemented to the medium (39, 44), which may be high enough for stimulation of progenitor cells to differentiate into SMCs. This is supported by our findings of PDGFR-beta siRNA knockdown studies showing an inhibited role of SMC differentiation. Another explanation for these results is cross talk between integrins and growth factor receptors. Many studies demonstrated that cross talk between cell adhesion receptors and growth factor receptors is an important molecular determinant in providing specificity for signaling during normal development and/or during pathological processes. Although integrins and growth factor receptors can independently propagate intracellular signals, the synergy of signals provided by the ECM and growth factors appears to regulate complex processes, including blood vessel development during embryogenesis as well as tumor growth/metastasis and angiogenesis in the adult (14). Thus it could be interesting to further study the molecular mechanisms for cross talk between integrins and growth factor receptors and the functional role in SMC differentiation.

Integrins have been identified as an important regulator of progenitor cell homing (5, 12), proliferation (2, 10, 36, 41), and differentiation (52) by influencing the balance between stem cell renewal and differentiation (47). Collagen-related integrins include {alpha}1beta1, {alpha}2beta1, {alpha}3beta1, {alpha}10beta1, and {alpha}11beta1 (29), in which integrin {alpha}1beta1 is abundant on SMCs. In the present study, we used collagen IV as ECM to activate the integrin signaling pathway and mediate SMC differentiation. High levels of integrin {alpha}1, {alpha}v, beta1, and beta3 were observed on both Sca-1+ progenitor cells and esSMCs, indicating their possible role for SMC differentiation. In line with this hypothesis, inhibition of the integrin pathway by blockade with RGD peptides and blocking antibodies against integrin {alpha}1, {alpha}v, and beta1 markedly inhibited SMC differentiation. Concomitantly, evidence from other groups indicates that loss of beta1 integrin function results in a retardation of myogenic differentiation of ES cells (31) and that {alpha}v integrins play an important role in myofibroblast differentiation (28).

One interesting finding in the present study is that collagen IV has a crucial role in stem cell differentiation in non-collagen IV-coated plates, i.e., collagen I and fibronectin. We demonstrated that collagen IV produced by the cells has a functional role in SMC differentiation, as verified by using a collagen IV-specific neutralizing antibody. It is known that, after withdraw of LIF, which is a key factor to maintain the undifferentiated status of ES cells in in vitro culture, ES cells can spontaneously differentiate into progenitor cells and/or mature cells. Our findings can provide, at least in part, the reasons why spontaneous differentiation occurs in some culture conditions, e.g., autocrine production of collagen IV.

Aiming to identify signaling pathways involved in SMC differentiation, we examined the activation of downstream signal transducers of integrins, including FAK, paxillin, PI 3-kinase, and MAPKs. After binding of ECM, integrin conformation is altered, and, subsequently, heterodimers can participate in events critical for organization of the cytoskeleton and other intracellular signaling events that might be important for cell differentiation. In the present study, we found that the level of FAK and paxillin phosphorylation was increased in esSMC during progenitor differentiation. RGD peptide G4391 and antibodies against integrin {alpha}1, beta1, and {alpha}v markedly or completely inhibited FAK and paxillin phosphorylation, leading to inhibition of SMC differentiation.

FAK activation is mediated primarily by autophosphorylation of tyrosine 397 that creates a binding site for PI 3-kinase (6). We hypothesized that PI 3-kinase has a functional role in the integrins-FAK/paxillin signaling pathway mediating SMC differentiation. We demonstrated that the protein level of PI 3-kinase (p85{alpha}) was increased under our experimental conditions. PI 3-kinase-specific inhibitor LY-294002 and siRNA significantly inhibited SMC differentiation. Meanwhile, a previous study showed that PI 3-kinase activates MAPK through its activity as a protein kinase (3), and PI 3-kinase is required for integrin-stimulated Akt and Raf-1/MAPK pathway activation(25). Interestingly, we observed that PI 3-kinase activity was directly related to the activation of ERK and JNK. Inhibition of JNK or ERK activation, but not p38MAPK, resulted in prevention of SMC differentiation. These results strongly suggested PI 3-kinase-ERK/JNK signaling pathways mediate SMC differentiation.

In summary, our results indicate that Sca-1+ cells derived from ES cells can differentiate into SMCs, SMC differentiation from Sca-1+ progenitor cells was mediated by the collagen IV-integrin ({alpha}1, beta1, and {alpha}v)-FAK/paxillin-PI 3-kinase-MEK-ERK/JNK and PDGFRbeta signaling pathways, and that collagen IV has a functional role in SMC differentiation. Our findings may hold therapeutic possibilities for cell therapy for certain types of vascular diseases and tissue engineering.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from the Oak Foundation and British Heart Foundation.


   ACKNOWLEDGMENTS
 
We thank Dr. Neil Roberts and Dr. Evelyn Torsney for critical reading of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Q. Xiao, Dept. of Cardiac and Vascular Sciences, St. George's, Univ. of London, Cranmer Terrace, London SW17 0RE, UK (e-mail: qingzhongxiao{at}hotmail.com)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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