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Keywords:

  • Differentiation;
  • Toll-like receptors;
  • Proliferation;
  • Human adipose stromal cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Adult mesenchymal stem cells (MSCs) are promising tools for such applications as tissue engineering and cellular therapy. It is not clear how stem cells exposed to unfavorable conditions (e.g., hypoxia or inflammation) respond to signals of danger after in vivo transplantation. Toll-like receptors (TLRs) play a major role in the immune system, participating in the initial recognition of microbial pathogens and pathogen-associated components. This study was designated to determine the role of TLRs in human MSCs. Reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry analysis demonstrated that MSCs derived from human adipose tissue and bone marrow express TLR-1, TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, and TLR-9. We investigated induction of the differentiation and proliferation of human adipose tissue stromal cells (hADSCs) by TLR agonists, including flagellin, peptidoglycans (PGN), lipopolysaccharide (LPS), the synthetic double-stranded RNA analog poly(I:C), and synthetic CpG oligodeoxydinucleotide (CpG-ODN). None of these agonists, except ODN, affected the proliferation of hADSCs. LPS and PGN increased osteogenic differentiation, but CpG-ODN decreased it. Poly(I:C) itself did not affect adipogenic or osteogenic differentiations, but exerted a synergistic effect on LPS- or PGN-induced osteogenic differentiation. RT-PCR analysis demonstrated that LPS and PGN induce osteogenic markers in hADSCs. TLR agonists affected the expression of chemokines and cytokines differentially. Furthermore, hADSCs affected the expression of specific TLRs in vitro under hypoxic conditions. These data provide evidence of a nonimmune role for TLR signaling on MSCs and may provide clues to the behavior of transplanted MSCs in vivo.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Toll-like receptors (TLRs), which are broadly distributed on cells throughout the immune system [1, 2], are the best-studied immune sensors of invading pathogens. TLR activation is essential for inducing the immune response and enhancing adaptive immunity against pathogens [3, 4]. Members of the TLR family are also involved in the pathogenesis of autoimmune, chronic inflammatory, and infectious diseases [5]. Thirteen mammalian TLR-1 analogs have been identified (10 in humans and 12 in mice) that recognize a wide variety of pathogen-associated molecular patterns in bacteria, viruses, and fungi, as well as certain host-derived molecules [6]. For example, TLR-2 recognizes bacterial lipoproteins, peptidoglycans (PGN), and lipoteichoic acids from Gram-positive bacteria; TLR-3 recognizes virus-derived double-stranded RNA and its DNA analog [poly(I:C)]; TLR-4 recognizes lipopolysaccharides (LPSs) from Gram-negative bacteria; TLR-5 recognizes bacterial flagellin; and TLR-9 recognizes the CpG motif of bacterial DNA. TLR-7 and TLR-8, which are phylogenetically close relatives and form an evolutionary cluster with TLR-9 [7], have recently been found to mediate recognition of single-stranded RNA [8, 9] and serve as receptors for small synthetic guanosine-based antiviral molecules such as loxoribine [10]. TLRs are also known to form complexes that confer a greater degree of specificity. For example, heterodimers of TLR-1/2 recognize triacetylated bacterial lipopeptides, whereas TLR-2/6 recognize diacetylated Mycoplasma lipopeptides [11]. A new member of the TLR family, TLR-11, has recently been identified in mice and responds specifically to uropathogenic bacteria [12]. The natural ligands for TLR-10 have not been identified. Recent studies revealed that endogenous TLR ligands such as heat shock protein and high mobility group box 1 are released from necrotic cells [13].

TLRs are type I transmembrane glycoproteins containing an extracellular domain composed of numerous leucine-rich repeats and an intracellular region containing a terminal inverted repeats (TIR) homology domain [14, 15]. The TIR domains interact with several TIR domain-containing adapter molecules (MyD88, TIRAP, TRIF, and TRAM) that activate a cascade of events resulting in transcription factor induction [16]. The common signaling feature among all of the TLRs is activation of the transcription factor nuclear factor-κB, which has been implicated in controlling the expression of inflammatory cytokines and maturation molecules. A subset of TLRs induces the production of type I interferons [17, 18], which mediate antiviral, growth inhibitory, and immunomodulatory responses. Recently, a novel nonimmune role for TLRs has been reported. Rakoff-Nahoum et al. [19] showed that TLR signaling can maintain epithelial homeostasis through proliferation and tissue repair after direct injury to the epithelium. Hasan et al. [20] reported that TLR signaling stimulates cell cycle entry and progression in fibroblasts. The discovery of TLR-induced proliferation and recent work describing a role for TLR in tissue repair prompted us to investigate the potential direct link between TLR signaling and stem cell functions.

Mesenchymal stem cells (MSCs) are adult progenitor cells in the bone marrow, peripheral blood, and adipose tissues which are able to differentiate into several lineages, including chondrocytes, osteoblasts, tendinocytes, myocytes, endothelial cells, neural cells, and adipocytes [21]. Because of their differentiation potential and the ease of expansion, they are studied largely for their use in tissue engineering for bone and cartilage repair and cell therapy for cardiovascular and central nervous system disorders. If transplanted stem cells are exposed to unfavorable conditions such as hypoxia or cytokines released from necrotic or inflammatory cells, changes in their characteristics can be expected. In fact, it has been reported that the differentiation potential and cytokine expression of MSCs are altered under hypoxic conditions [22]. The expression profiles for TLRs, their functions, and signaling pathways have been elucidated in various types of mammalian immune cells [18, 23, 24], but limited information is available for adult stem cells.

The aim of our study was to determine whether human adipose tissue stromal cells (hADSCs) express TLRs and whether they respond to TLR ligands. The results showed that hADSCs and human bone marrow stromal cells (hBMSCs) express multiple TLRs and respond to TLR ligands differentially. Furthermore, their expression was altered by exposure of hypoxia. These observations will provide the background information needed to understand the fate of stem cells that are transplanted into injured tissues.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Reagents

LPS (Escherichia coli, O55:B5), the synthetic double-stranded RNA analog poly(I:C), and PGN (Staphylococcus aureus) were purchased from Sigma-Aldrich (St. Louis, http://www.sigmaaldrich.com) and Fluka (Sigma-Aldrich). Flagellin (Salmonella typhimurium) and synthetic oligodeoxydinucleotides (ODNs) (2006, 2216) were purchased from InvivoGen (San Diego, http://www.invivogen.com).

Cell Culture

hBMSCs and hADSCs were cultured as described by Lee et al. [25]. All protocols involving human subjects were approved by the Institutional Review Board of the Pusan National University. To isolate hADSCs, adipose tissue samples were washed with phosphate-buffered saline (PBS) and digested at 37°C for 30 minutes by 0.075% type I collagenase. Enzyme activity was neutralized with α-modified Eagle's medium (α-MEM) containing 10% fetal bovine serum (FBS) and centrifuged at 1,200g for 10 minutes to obtain a pellet. The pellet was incubated overnight at 37°C under 5% CO2 in a control medium (α-MEM, 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin). After incubation, the tissue culture plates were washed to remove residual nonadherent cells and were maintained at 37°C under 5% CO2 in the control medium. Mononuclear cells from bone marrow were separated by centrifugation in a Ficoll-Hypaque gradient (density = 1.077 g/cm3; Sigma-Aldrich), suspended in α-MEM containing 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin, and seeded at a concentration of 1 × 106 cells per cm2. Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2. When the monolayer of adherent cells reached confluence, they were trypsinized (0.25% trypsin; Sigma-Aldrich), resuspended in α-MEM containing 10% FBS, and subcultured at a concentration of 2,000 cells per cm2. For this experiment, we used third to fifth passes of MSCs.

Flow Cytometric Analysis

hADSCs were cultured in control medium for 72 hours prior to analysis. Flow cytometry was performed on a FACScan argon laser cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). Cells were harvested in 0.25% trypsin/EDTA and fixed for 30 minutes in ice-cold 4% formaldehyde. After fixation, cells were washed in flow cytometry buffer (FCB) (1 × PBS, 2% FBS, 0.05% sodium azide). Cell aliquots (1 × 106 cells) were incubated in FCB containing 20 μl of monoclonal antibodies to fluorescein isothiocyanate (FITC)-conjugated anti-human CD3, phycoerythrin (PE)-conjugated anti-human CD19 (Becton, Dickinson and Company), FITC-conjugated anti-human TLR-2, PE-conjugated anti-human TLR-3, PE-conjugated anti-human TLR-4, and PE-conjugated anti-human TLR-9 (eBiosciences, San Diego, http://www.ebiosciences.com). For the detection of TLR-3 and TLR-9, the cells were permeabilized with 0.1% Triton X-100 before incubation with the antibody. Between each step, cells were washed with PBS plus 0.3% BSA. As control, cells were stained with PE-conjugated or FITC-conjugated anti-mouse immunoglobulin G1 isotype control antibodies (eBiosciences). Three samples from different individuals were used in this test.

Induction of Differentiation

Adipogenic differentiation was induced by culturing MSCs for 2 weeks in an adipogenic medium (10% FBS, 1 μM dexamethasone (Decadron; Merck & Co., Inc., Whitehouse Station, NJ, http://www.merck.com), 100 μg/ml 3-isobutyl-1-methylxanthine, 5 μg/ml insulin, and 60 μM indomethacin in α-MEM) and assessed by the use of an oil red O stain as an indicator of intracellular lipid accumulation. To obtain quantitative data, 1 ml of isopropyl alcohol was added to the stained culture dish. Osteogenic differentiation was induced by culturing cells in an osteogenic medium (OM) (10% FBS, 0.1 μM dexamethasone, 10 mM β-glycerophosphate, and 50 μM ascorbic acid in α-MEM), and the degree of extracellular matrix calcification was estimated using alizarin red S stain. Osteogenic differentiation was quantified by measuring the total signal of each well that represents a stained area and its intensity by an image analysis program (Image Gauge, version 3.0; FUJIFILM Corporation, Tokyo, http://www.fujifilm.com). Measurements were done in duplicate in each experiment, and the experiments were repeated using tissue from three different donors. To determine osteognic gene expression during differentiation, we used reverse transcriptase-polymerase chain reaction (RT-PCR) analysis with primers (glyceraldehyde-3-phosphate [GAPDH] 5′-TCCATGACAACTTTGGTATCG-3′, 5′-TGTAGCCAAATTCGTTGTCA-3′, Runx2 5′-CTCACTACCACACCTACCTG-3′, 5′-TCAATATGGTCGCCAAACAGATTC-3′, alkaline phosphatase 5′-TGAAATATGCCCTGGAGC-3′, 5′-TCACGTTGTTCCTGTTTAG-3′, osteopontin 5′-TTGCAGTGATTTGCTTTTGC-3′, 5′-ACACTATCACCTCGGCCATC-3′, and bone morphogenetic protein [BMP]2 5′-CCA CCA TGA AGA ATC TTT GG-3′, 5′-CCA CGT ACA AAG GGT GTC TC-3′). All primer sequences were determined by comparison with established GenBank sequences. Duplicate PCRs were amplified using the primer's designed GAPDH as a control to assess PCR efficiency and for subsequent analysis by agarose gel electrophoresis. Noninduced hADSCs were examined for use as a negative control.

Evaluation of Cell Proliferation and Viability

To determine the proliferation rate, cells were detached using Hanks' balanced salt solution containing 0.5% trypsin and 0.02% EDTA. hADSCs were plated at a density of 1,000/cm2 in a 60-well plate, and various concentrations of TLR agonists were added the day after plating. After 48 hours, the cells were trypsinized and stained with 0.4% trypan blue (Sigma-Aldrich). The total cell number and the proportion of dead cells were measured by hemocytometer. Cell death was determined by the presence of cytoplasmic trypan blue. This experiment was performed in triplicate. The effect of CpG-ODN on cell cycle distribution was determined by flow cytometric analysis of DNA content of nuclei of cells after staining with propidium iodide. hADSCs (106 cells) were cultured for 24 hours with CpG-ODN 2006 (1 μg/ml). The cells were washed with PBS and fixed in 70% ethanol overnight at 4°C. The cells were then treated with 80 μg/ml RNaseA and 50 μg/ml propidium iodide for 30 minutes and analyzed using a FACScan argon laser cytometer (Becton, Dickinson and Company).

Analysis of TLR and Cytokine Expression in hMSCs by RT-PCR

To determine TLR expression in MSCs, total cellular RNA was isolated from hADSCs and hBMSCs. To examine the effects of TLR agonists on cytokine expression, hADSCs were cultured for 24 hours with various TLR agonists and total RNA was isolated. TLR agonists were used at the following concentrations: PGN, 10 μg/ml; poly(I:C), 25 μg/ml; LPS, 10 μg/ml; flagellin, 5 μg/ml; and CpG-ODN 2006, 1 μg/ml. Total RNAs were reverse-transcribed using conventional protocols. All primers for PCR amplification were designated by Primer 3.0 from established GenBank sequences (supplemental online Tables 1 and 2). The expression of various transcripts was assessed by PCR amplification, using a standard protocol as previously described. Amplified products were analyzed by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining. Semiquantitative analysis of transcript abundance was assessed relative to GAPDH expression using Image analysis software (Image Gauge, version 3.0).

Culture of hADSCs at Hypoxic Condition

hADSCs were grown for 48 hours in a hypoxic incubator (ASTEC CO2 water jacket incubator; ASTEC, Fukuoka, Japan, http://www.astec-bio.com) at an atmosphere of 5% CO2, 2% O2, and balance N2. The change in TLR genes in hypoxic hADSCs was analyzed using RT-PCR as described above.

Western Blot Analysis

Confluent hADSCs were treated under appropriate conditions and then lysed, and their protein content was determined using a protein assay kit (Bio-Rad, Hercules, CA, http://www.bio-rad.com). Proteins were loaded on a 10% SDS-polyacrylamide gel and electrotransferred onto nitrocellulose membranes (Hybond-ECL; GE Healthcare, Little Chalfont, Buckinghamshire, U.K., http://www.gehealthcare.com), then probed with p44/42 MAP kinase antibody (total p44/42 MAP kinase [Erk1/Erk2]; Cell Signaling Technology, Inc., Danvers, MA, http://www.cellsignal.com) or antibody for phosphorylated extracellular signal-regulated kinase (ERK) (Antiphospho-ERK [Thr202/204]; Cell Signaling Technology, Inc.). Immunoreactive bands were detected using anti-mouse peroxidase-conjugated secondary antibody (GE Healthcare) and visualized by enhanced chemiluminescence (ECL detection kit; GE Healthcare).

Statistical Analysis

Comparisons of multiple groups were performed by analysis of variance with Bonferroni correction for multiple comparisons.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Expression of TLRs in MSCs Isolated from Bone Marrow and Adipose Tissue

PCR primers specific to TLR-1 to TLR-10 were designed to detect the expression of TLRs from total RNA in hMSCs. RT-PCR analysis showed that hMSCs express TLR-1, TLR-2, TLR-3, TLR-4, TLR-6, and TLR-9 (Fig. 1). The most highly expressed TLRs were TLR-2, TLR-3, TLR-4, and TLR-6; the lowest rates detected were for TLR-1, TLR-5, and TLR-9. To determine whether TLR transcripts were derived from contaminating B or T lymphocytes and leukocytes, we analyzed expressions of CD3 as a pan T lymphocyte marker, CD19 as a pan B lymphocyte marker, and CD45 as a pan leukocyte marker by flow cytometry in hADSCs. hADSCs did not express CD3, CD19, and CD45, excluding B- and T-lymphocyte and leukocyte contamination (Fig. 1B). Flow cytometry analysis of TLR-2, TLR-3, TLR-4, and TLR-9 showed their homogenous expression in hADSCs (Fig. 1C).

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Figure Figure 1.. Expression of TLRs in hMSCs. (A): Total RNAs were isolated from hMSCs and synthesized with reverse transcriptase. Polymerase chain reaction products for TLR-1 to TLR-10 were separated by agarose gel electrophoresis. GAPDH was amplified as a positive control. (B): Assessment of lymphocyte and leukocyte contamination in hADSCs by fluorescence-activated cell sorting analysis. hADSCs were stained with anti-CD3, CD19, and CD45 antibodies and PE-conjugated or fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin G1 isotype control antibodies. A representative histogram of three separate experiments was presented. (C): Flow cytometry detection of TLRs in hADSCs. hADSCs were stained with anti-TLR-2, -TLR-3, -TLR-4, and -TLR-9 antibodies. Abbreviations: GAPDH, glyceraldehyde-3-phosphate; hADSC, human adipose tissue stromal cell; hBMSC, human bone marrow stromal cell; hMSC, human mesenchymal stem cell; PE, phycoerythrin; TLR, Toll-like receptor.

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Effect of TLR Agonists on Proliferation of hADSCs

To determine the role of TLRs in the proliferation of hADSCs, we first examined the effects of TLR agonists (10 μg/ml LPS, 10 μg/ml PGN, 25 μg/ml poly(I:C), 1 μg/ml CpG-ODN 2006 and 2216, and 5 μg/ml flagellin) on the proliferation of hADSCs (Fig. 2A). CpG-ODN 2006 and 2216 (1 μg/ml) inhibited hADSC proliferation, whereas the other agonists had no effect on it. We determined the dose-response relationship of the CpG-ODN effect on hADSC proliferation. CpG-ODN 2006 and 2216 inhibited hADSC proliferation in a dose-dependent manner (Fig. 2B). The trypan blue exclusion study showed that CpG-ODN does not induce cell death within the concentration range tested in this experiment. Flow cytometric analysis of cell cycles showed that CpG-ODN 2006 induced G1 arrest of hADSCs (Fig. 2C).

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Figure Figure 2.. Effect of TLR agonists on the proliferation of hADSCs. The day after plating hADSCs (1,000 cells per cm2), various TLR agonists (10 μg/ml PGN, 25 μg/ml poly(1:C), 10 μg/ml LPS, 5 μg/ml flagellin, and 1 μg/ml CpG-ODN) (A) and CpG-ODNs (0.5, 1, and 2 μg/ml) (B) were added. Proliferation of hADSCs was determined by direct cell counting using a hemocytometer 48 hours after the addition of TLR agonists. The percentage of viable cells was determined by trypan blue staining. Data represent mean ± SEM of three different experiments. ∗ p < .05 compared with control. (C): Effect of CpG-ODN 2006 on hADSC cell cycle distribution. A representative histogram from control and CpG-ODN 2006-treated (1 μg/ml for 24 hours) hADSCs was shown. Abbreviations: hADSC, human adipose tissue stromal cell; LPS, lipopolysaccharide; ODN, oligodeoxydinucleotide; PGN, peptidoglycans; TLR, Toll-like receptor.

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Effect of TLR Ligands on Differentiation of hADSCs

To determine whether TLR agonists affect the osteogenic and adipogenic differentiations of hADSCs, we added TLR agonists to the differentiation media. LPS and PGN strongly stimulated the osteogenic differentiation of hADSCs in a dose-dependent manner, whereas CpG-ODN 2006 and 2216 inhibited it. Poly(I:C) and flagellin had no effect on it (Fig. 3A). In the case of adipogenic differentiation, PGN inhibited it significantly (Fig. 3A), but the other agonists did not affect it (data not shown). To further confirm whether LPS and PGN increased osteogenic differentiation, we determined the expression of osteogenic differentiation markers by means of RT-PCR analysis. When LPS, PGN, or CpG-ODN 2006 was added to the osteogenic differentiation media, LPS and PGN increased the expression of alkaline phosphatase (an osteogenic marker), osteopontin, and BMP2. In CpG-ODN 2006-treated cells, osteopontin and runx2 expression at the 10th day after differentiation decreased, compared with control (Fig. 4).

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Figure Figure 3.. Effect of TLR agonists on osteogenic and adipogenic differentiations of hADSCs. Cells were grown to confluence and then induced to osteogenic or adipogenic differentiations in differentiation media containing TLR agonists. (A): Osteogenic differentiation was determined by calcification deposits within the cell monolayer, which were stained with alizarin red S. Adipogenic differentiation was determined with oil red O stain as an indicator of intracellular lipid accumulation. (B): The quantitation of osteogenic and adipogenic differentiations was performed by determination of density and area of alizarin red S staining with an image analysis program (Image Gauge version 3.0) and measurement of optical density in isopropanol extract of oil red O staining, respectively. Data were presented as a percentage of control (mean ± SEM, n = 3). ∗ p < .05 compared with control. Abbreviations: AM, adipogenic medium; Cont, control; FN, flagellin; LPS, lipopolysaccharide; ODN, oligodeoxydinucleotide; OM, osteogenic medium; PGN, peptidoglycans.

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Figure Figure 4.. RT-PCR analysis of osteogenic genes. hADSCs were grown to confluence in CM, and osteogenic differentiation was induced by DM with or without TLR agonists. Total RNA were isolated at the 5th and 10th days after induction of differentiation. (A): Osteogenic genes were determined by RT-PCR. GAPDH was amplified as a positive control. (B): Quantitation of PCR products. Quantity of amplified products was analyzed by an image analyzer. Data represent mean ± SEM of the relative ratio to GAPDH signal of the corresponding samples (n = 3). ∗ p < .05 compared with control. Abbreviations: AP, alkaline phosphatase; BMP, bone morphogenetic protein; CM, control medium; DM, differentiation media; GAPDH, glyceraldehyde-3-phosphate; hADSC, human adipose tissue stromal cell; LPS, lipopolysaccharide; ODN, oligodeoxydinucleotide; OPN, osteopontin; PCR, polymerase chain reaction; PGN, peptidoglycans; RT-PCR, reverse transcriptase-polymerase chain reaction; TLR, Toll-like receptor.

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LPS- or PGN-Induced Osteogenic Differentiation in hADSCs Was Accompanied with Increase of ERK Activation

It has been reported that activation of ERK plays an important role in the osteogenic differentiation of MSCs [26]. Therefore, we examined ERK activation for 12 days during treatment with osteogenic medium in the presence or absence of LPS, PGN, and CpG-ODN 2006. ERK activity in OM-treated cultures was determined by immunoblot analysis using phosphospecific MAP kinase antibody. As shown in Figure 5, OM did not have an effect on ERK activation up to day 3 of treatment. However, a robust and sustained activation of ERK was observed at day 7. The maximal activation was observed at day 7, and ERK activity declined to basal levels after day 12. Treatment with LPS or PGN increased OM-induced ERK activation at day 7, whereas CpG-ODN 2006 inhibited it.

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Figure Figure 5.. ERK activation by TLR agonists in hADSCs. hADSCs were grown to confluence, and osteogenic differentiation was induced by the treatment of differentiation media containing TLR agonists (10 μg/ml PGN, 10 μg/ml LPS, and 1 μg/ml CpG-ODN 2006). Phosphorylation of ERK was determined by Western blotting of the cell lysates with anti-phospho-ERK1/2 antibody, and the amounts of ERK were probed by anti-ERK antibody to confirm equal loading. Representative data from three independent experiments are shown. Abbreviations: ERK, extracellular signal-regulated kinase; LPS, lipopolysaccharide; ODN, oligodeoxydinucleotide; PGN, peptidoglycans; TLR, Toll-like receptor.

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TLR Agonist-Induced Changes in Cytokine Expression

We analyzed the effects of TLR agonists on the expression of cytokines and chemokines in hADSCs using RT-PCR. The total RNA was isolated from hADSCs treated with TLR agonists for 24 hours. Upon stimulation, the patterns of alterations in cytokine and chemokine expression and the magnitude of their expression varied by TLR agonist.

The TLR agonists to be examined in this study increased monocytes chemotactic protein (MCP)-2, granulocyte chemotactic protein-2 (GCP-2), and interleukin (IL)-1β expression. LPS, poly(I:C), flagellin, and CpG-ODN 2006 increased macrophage inflammatory protein-3 α (MIP-3α) and IL-6, but PGN did not. PGN, LPS, poly(I:C), and flagellin increased tumor necrosis factor (TNF)-α and IL-12 expression, but CpG-ODN 2006 did not. Poly(I:C) and LPS exposure resulted in increased expression of MCP-1, but CpG-ODN 2006 and PGN had no effect on it (Fig. 6).

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Figure Figure 6.. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of cytokine mRNA levels by stimulation Toll-like receptor (TLR) agonists. hADSCs were stimulated with various TLR agonists for 24 hours. (A): Cytokine mRNA levels in control hADSCs and hADSCs after stimulation with TLR agonists were determined by RT-PCR. GAPDH was amplified as a positive control. TLR agonists were used at the following concentrations: PGN (10 μg/ml), poly(I:C) (25 μg/ml), LPS (10 μg/ml), flagellin (5 μg/ml), and CpG-ODN 2006 (1 μg/ml). (B): Quantitation of polymerase chain reaction products. Amounts of amplified products were quantitated by an image analyzer. Data represent the mean ± SEM of the relative ratio to GAPDH signal of the corresponding samples (n = 3). ∗ p < .05 compared with control. Abbreviations: GAPDH, glyceraldehyde-3-phosphate; GCP, granulocyte chemotactic protein; hADSC, human adipose tissue stromal cell; IL, interleukin; LPS, lipopolysaccharide; MCP, monocytes chemotactic protein; MIP, macrophage inflammatory protein; ODN, oligodeoxydinucleotide; PCR, polymerase chain reaction; PGN, peptidoglycans; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor.

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Effect of Hypoxia on the Expression of TLRs in hADSCs

hADSCs can be transplanted into tissue injured by ischemia under hypoxic conditions. To determine whether hypoxia can affect the expression of TLRs in hADSCs, we cultured hADSCs at 2% O2 and then isolated the total RNA 3 days after hypoxia. Exposure to hypoxic conditions significantly increased the expression of TLR-1, TLR-2, TLR-5, TLR-9, and TLR-10 (Fig. 7).

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Figure Figure 7.. Changes of TLR expression in human adipose tissue stromal cell (hADSCs) under hypoxic culture condition. hADSCs were grown to confluence and then cultured for 72 hours in a hypoxic incubator at an atmosphere of 5% CO2, 2% O2, and balance N2. (A): Total RNAs were isolated from hADSCs grown under normoxic or hypoxic culture condition, and changes in expression of TLRs were determined by reverse transcriptase-polymerase chain reaction. (B): Quantitation of polymerase chain reaction products. Quantitation of amplified products was done by an image analyzer. Data represent mean ± SEM of the relative ratio to GAPDH signal of the corresponding samples (n = 3). ∗ p < .05 compared with control (Cont). Abbreviations: GAPDH, glyceraldehyde-3-phosphate; TLR, Toll-like receptor.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

TLRs are known to be expressed differentially in tissues and cellular subsets of leukocytes [1, 27, 28]. In the present study, we examined the expression profiles for TLRs in hADSCs and hBMSCs detected by RT-PCR amplification using primers specific for human TLR-1 to TLR-10. PCR amplification resulted in intensive DNA bands for TLR-2, TLR-3, TLR-4, and TLR-6 followed by a less intense band for TLR-1, TLR-5, and TLR-9, indicating that these receptors are expressed in hADSCs and hBMSCs. Amplification of TLR transcripts from contaminating lymphocytes and leukocytes can be excluded by the flow cytometry findings that hADSCs do not express B- and T-lymphocyte markers (CD3 and CD19) and leukocyte marker (CD45) and showed homogenous expression of TLRs.

To determine the functional role of TLRs in hADSCs, LPS (agonist for TLR-4), PGN (agonist for TLR-2), CpG-ODN DNA (oligodeoxynucleotide 2216 and 2006 agonists for TLR-9), poly(I:C) (agonist for TLR-3), and flagellin (agonist for TLR-5) were tested for their ability to activate hADSCs. Activation of TLR-9 by CpG-ODN decreased hADSC proliferation, whereas the other TLR ligands had no effect. In recent studies, investigators have shown that stimulation of TLR-5 expressed in Rat1 cells with flagellin induces the proliferation of serum-starved fibroblasts [20] and that a TLR-9 agonist inhibited proliferation of a colon cancer cell line [29]. These findings support the hypothesis that TLR activation may affect proliferating ability of transplanted hADSCs in vivo.

In this study, we found that TLR agonists induced hADSC differentiation according to subtype. LPS and PGN increased osteogenic differentiation, whereas CpG-ODN 2006 and 2216 decreased osteogenic differentiation. Poly(I:C) itself did not affect differentiation. These results indicate that hMSCs express various TLRs and that their activation induces various types of responses that affect the therapeutic efficacy of stem cells. Although the signaling mechanisms for LPS- or PGN-induced osteogenic differentiation are not completely solved, our data in this study indicated that ERK activation and BMP2 expression can be involved in the effect of LPS, PGN, and CpG-ODN on osteogenic differentiation in hADSCs.

Cytokine analysis by RT-PCR showed that TLR agonists affect the expression of GCP-2, MCP-1 and MCP-2, MIP-3α, IL-12, TNF-α, and IL-1β. Some of these cytokines have been reported to be involved in alterations of stem cell function as well as recruitment of inflammatory cells or immune cells (e.g., dendritic cells [DCs]) by injured tissues [30]. MCP-1 induced the migration of hBMSCs to injured tissues [31, 32]. GCP-2 and MIP-3 have been reported to be involved in angiogenesis [33, 34]. IL-12 plays an important role in DC activation and killer T-cell activity [35]. Immature DCs derived from CD34 hematopoietic progenitor cells migrate most vigorously in response to MIP-3, but also to MIP-1 and RANTES (regulated on activation normal T cell expressed and secreted). MIP-3/CCL20 is a unique functional ligand for the recruitment of a distinct population of CCR6-expressing immature DCs to tissue for subsequent antigen presentation [36, 37].

Some cytokines (e.g., TNF-α, IL-1β, or IL-12) have been shown to affect osteoclastogenesis or bone formation [38, 39]. However, we could not explain the TLR agonist-induced effect on osteogenic differentiation in which an increase in TNF expression did not correlate with the induction of osteogenic differentiation by TLR ligands. ODNs that inhibited osteogenic differentiation decreased TNF-α expression, whereas PGN and LPS, which increased osteogenic differentiation, increased TNF-α expression.

The finding that functional TLRs are present at the stem cell stage raises the possibility that TLR ligands influence numbers and characteristics of stem cells in transplanted animals. In this study, we also found that culturing hADSCs under hypoxic conditions resulted in increased expression of TLR-1, TLR-2, TLR-5, and TLR-9. Altered hADSC activity induced by TLR ligands and changes in TLR expression under hypoxic conditions can play a significant role in several biologically relevant situations. When stem cells are transplanted for cell replacement therapy, the transplanted cells are often exposed to unfavorable conditions such as hypoxia or inflammation. It has been reported that endogenous TLR ligands are present in normal tissues [40] and can be released from damaged tissues [13]. TLRs on transplanted MSCs sense “danger signals” represented by injured tissues, which, in turn, may affect the tissue repair processes by transplanted stem cells. Roles of TLRs on differentiation of progenitors have been reported in innate immune system replenishment by hematopoietic progenitors [41] and in regeneration of intestinal epithelia [42]. Further investigation of these processes will clarify how signals delivered via TLRs influence stem cell behavior.

Taken together, our findings demonstrate that hMSCs express functional TLRs and that the expression of these receptors may be altered during cell activation. Activation of hADSCs with TLR agonists triggers the increased expression of a number of chemokines and cytokines. We speculate that the effect of TLR activation on MSCs may affect the in vivo fate of transplanted cells and that manipulatioin of TLR signals may provide new ways for modulation of stem cell function.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

This work was supported by the Medical Research Council program of MOSF/Korean Science and Engineering Foundation (R13-2005-009).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information
FilenameFormatSizeDescription
Supp_Table_1.pdf42KSupplemental Table 1
Supp_Table_2.pdf50KSupplemental Table 2

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