Stem Cell Rev and Rep DOI 10.1007/s12015-014-9498-z
Divergent Levels of LBP and TGFβ1 in Murine MSCs Lead to Heterogenic Response to TLR and Proinflammatory Cytokine Activation Sarit Levin & Meirav Pevsner-Fischer & Sivan Kagan & Hila Lifshitz & Ada Weinstock & Diana Gataulin & Gilgi Friedlander & Dov Zipori
# Springer Science+Business Media New York 2014
Abstract The outstanding heterogeneity of stem cell populations is a major obstacle on the way to their clinical application. It is therefore paramount to identify the molecular mechanisms that underlay this heterogeneity. Individually derived bone marrow mesenchymal stromal cells (MSCs) preparations, studied here, diverged markedly in various properties, despite of being all tripotent in their differentiation potential. Microarray analysis showed that MSC diversity is evident also in highly variable gene expression patterns. Differentially expressed genes were significantly enriched in toll-like receptors (TLRs) and differentiation pathways. Marked differences were observed in LPS binding protein (LBP) and transforming growth factor (TGF)β1 expression. These differences correlated with MSC functionality. Therefore, the possible contribution of these molecules to MSC diversity was examined. In the TLR signaling pathway, LBP levels predicted the ability of specific MSCs to secrete interleukin (IL)-6 in response to LPS. A relatively higher expression of TGFβ1 endowed MSCs with a capacity to respond to IL-1β by reduced Sarit Levin and Meirav Pevsner-Fischer contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s12015-014-9498-z) contains supplementary material, which is available to authorized users. S. Levin : M. Pevsner-Fischer : S. Kagan : H. Lifshitz : A. Weinstock : D. Gataulin : D. Zipori Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel 76100 G. Friedlander The Biological Services Unit, Weizmann Institute of Science, Rehovot, Israel 76100 D. Zipori (*) Department of Molecular Cell Biology, Weizmann Institute of Science, 234 Herzel St., Rehovot 7632700, Israel e-mail: [email protected] URL: http://www.weizmann.ac.il/mcb/Zipori/
osteogenic differentiation. This study thus demonstrates major diversity within MSC isolates, which appears early on following derivation and persists following long–term culture. MSC heterogeneity results from highly variable transcriptome. Differential expression of LBP and TGFβ1, along with other genes, in different MSC preparations, produces the variable responses to external stimuli. Keywords MSC . Heterogeneity . LBP . TGFβ1 . TLR
Introduction Mesenchymal stromal cells (MSCs) are adult progenitors that were originally obtained by expansion of the bone marrow (BM) plastic-adherent cell fraction [1, 2]. Their capacity to differentiate in vitro into multiple mesodermal lineages is used as a functional, retrospective defining criterion [1–3]. MSCs display a variety of functions, including the support and regulation of hematopoiesis in vivo and in vitro[4–6], regulation of immune responses [7–11], migration into injured tissues and participation in the repair of tissue damage [12–16]. The heterogeneity of BM-MSCs is a well-known phenomenon. Individual MSC preparations differ in morphology and in their growth rate and differentiation potentials [17]. A hierarchical model was proposed to explain the existence of MSC clones with divergent differentiation potential. Multi-potent mouse and rat MSCs were suggested to give rise to more restricted clones and to finally differentiate into mono-potent progenitor cells [18]. This process was suggested to be a nonrandom, single-step process in which multi-potential progenitors become exclusively restricted to a single lineage by particular culture conditions and inducers [19–21], but this view is still a matter of debate [22–24].
Stem Cell Rev and Rep
We and others have previously showed that cultured murine MSCs express toll-like receptors (TLR)s [25–32]. The activation of MSCs by TLR ligands induced interleukin-6 (IL6) secretion and NFкB nuclear translocation. Pam3Cys, a prototypic ligand for TLR2, induced proliferation of MSCs and regulated their differentiation [33]. It was also shown that TLR activation regulates human MSC differentiation, proliferation, immune regulation and migration [25–32]. However, there is a lack of consensus in relation to the mode by which MSCs respond to TLR ligands due to conflicting reports on various effects of TLR activation on MSCs. The present study aims to investigate the molecular basis for the heterogeneity in the response of MSCs to TLR activation and its origin. To exclude differences resulting form the position of the cells within the hierarchical lineage, we chose to examine tripotent MSCs only. It is shown herein that independent isolates of MSCs differ markedly in their functional properties. Transcriptome analysis of such divergent populations provides means to discover new regulatory pathways.
Materials and Methods Mice BALB/C and C57BL/6J mice were purchased from Harlan Olac. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of the Weizmann Institute of Science. Permit Number: 04400810-1. Cell Culture MSCs were grown in murine MesenCult Basal Media supplemented with 20 % murine mesenchymal supplement (StemCell Technologies, Vancouver, Canada), 60 μg/mL penicillin, 100 μg/mL streptomycin and 50 μg/mL kanamycin. MSCs from passages 4–6 and 14 to passage 20 were used in all experiments described. Reagents Escherichia coli O55:B5 LPS, peptidoglycan of Staphylococcus aureus, and polyinosinicpolycytidylic acid (Poly(I:C)) were purchased from Sigma (Rehovot, Israel). Pam3Cys was purchased from EMC microcollections (Tübingen, Germany). CpG—the phosphorothioaete oligonucleotides—was synthesized at the Oligonucleotide Synthesis Unit of the Weizmann Institute of Science (Rehovot, Israel). The oligonucleotide CpG contains 2 9-mer segments: 5′-TCCATAACGTTGCA AACGTTCTG-3′. TNFα, IL-1β and human TGFβ1 were
purchased from PeproTech/Cytolab (Rehovot, Israel). Polyclonal rabbit anti-ERK1/2, anti-JNK and anti-p38 were purchased from Sigma (Rehovot, Israel). Polyclonal rabbit anti– NFкB p65 was obtained from eBioscience (San Diego, CA). Polyclonal rabbit anti-Histone3 was obtained from Open biosystems (Huntsville, AL). Anti-phospho JNK and antiphospho ERK1/2 were purchased from Santa-Cruz Biotech (Santa Cruz, CA). Anti-phospho p38 was purchased from BioSource Invitrogen (Carlsbad, CA). BM Cell Extraction and MSC Production BM cells were obtained from femur and tibia of 6- to 8-weekold C57BL/6J mice, re-suspended in PBS with 1 % FBS and subjected to centrifugation. The cells were then seeded in 60mm plates containing MSC medium. Half of the medium was replaced every 3 days; upon confluence, the cells were removed using trypsin (0.05 % EDTA, 0.25 % trypsin; Biological Industries) and reseeded. For clone isolation, fresh BM cells from femur and tibia of 6–8 week-old BALB/C mice were seeded in 24-well plates (Falcon) at a concentration of 65 or 30×105 cells per well and propagated in MSC medium. Only wells containing single colonies were used for further experiments. Cell Sorting Cell sorting was conducted for MSC I as described in our previous work [33]. Evaluation of MSC Differentiation Osteogenesis Cells were seeded at a concentration of 3× 104cells/well in a 24-well plate. The next day, osteogenic medium containing 50 μg/mL L-ascorbic acid-2 phosphate, 10 mM glycerol 2-phosphate disodium salt, and 1×10−7 M dexamethasone (all from Sigma) were added, either with or without pam3cys, peptidoglycan, LPS, Poly(I:C), TNFα or IL-1β in the indicated concentrations. The cells were grown for 1 to 3 weeks with medium replacement twice a week. Osteogenic differentiation was detected by alizarin red staining. For alizarin red quantification, 0.5 N HCl and 5 % sodium dodecyl sulfate (SDS) were added to each well. Light absorbance by the extracted dye was measured in 405 nm. Real time-PCR Total RNA was isolated from confluent MSCs using TRI reagent (MRC, Cincinnati, OH). Contaminating DNA was removed using DNase treatment by TURBO DNA-free™ Kit (Ambion). First-strand cDNA synthesis was performed from 5 μg of total RNA by extension of oligo(dT) primers with 200 U Moloney murine leukemia virus reverse
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transcriptase (MMLV-RT; Promega, Madison, WI). Real-time PCR was performed on an ABI 7000 real-time PCR system, using SYBR green PCR master mix (Applied Biosystems, Lincoln Center, CA). The values for the specific genes were normalized to HPRT. Primer sequences used: LBP F: 5′TTTCATAGCCTGGAGATTCAGAACT-3′. LBP R: 5′TGGCCTGGGAGCAGCTT-3′. TGFβ1 F: 5′- TGAACC AAGGAGACGGAATACA-3′. TGFβ1 R: 5′- AAGAGCAG TGAGCGCTGAATC-3′. HPRT F: 5′- GCAGTACAGCCC CAAAATGG-3′. HPRT R: 5′- GGTCCTTTTCACCAGCAA GCT-3′. IL-6 ELISA MSCs at 1.8×104 cells/well were seeded in MSC medium in 96-well plates. Later (24 h), the medium was replaced with DMEM (Gibco/BRL, Gaithersburg, MD) and supplemented with 10 % FBS with or without all TLR ligands in the indicated concentrations. IL-6 concentration in culture media was determined by enzyme-linked immunosorbent assay (ELISA) for IL-6 (OptiEIA kit; BD Pharmingen, San Diego, CA) according to the manufacturer’s instructions. Standard curves were established using mouse recombinant IL-6. Western Blot Analysis For NF-kB detection, cell lysates were microcentrifuged at 950×g for separation of nuclear (pellet) and cytoplasmic (supernatant) fraction. The nuclear fraction was then lysed in a buffer containing 30 mM HEPES, 450 mM NaCl, 25 % glycerol, 0.5 mM EDTA, 12 mM MgCl2, 6 mM DTT, 1 mM PMSF, and protease and phosphatase inhibitors. Equal amounts of protein were loaded and electrophoresed on a 12 % SDS–polyacrylamide gel electrophoresis (SDSPAGE), transferred to nitrocellulose membranes, blocked and treated overnight with a rabbit polyclonal anti-Histone 3, rabbit polyclonal anti–ERK1/2, or rabbit polyclonal anti– NF-kB in PBS containing 0.05 % tween (PBST) with 1 % BSA. Following washing, the membranes were incubated with horseradish peroxidase (HRP)–conjugated anti–rabbit IgG antibody in PBST with 2.5 % skimmed milk. Electrochemiluminescence (ECL) detected the immunoreactive protein. For ERK1/2, JNK or P38 phosphorylation detection, equal amounts of whole cell lysates were loaded and electrophoresed on a 12 % SDS-PAGE, transferred to nitrocellulose membranes, blocked and treated overnight with either a anti-ERK1/2, anti-JNK, anti-p38, anti-phospho p38, anti-phospho JNK or anti-phospho ERK1/2 in PBS PBST with 1 % BSA. Following washing, the membranes were incubated with HRP–conjugated anti–rabbit or mouse IgG antibody in PBST with 2.5 % skimmed milk. ECL detected the immunoreactive protein.
Microarray Analysis Experiment was carried out using 100 ng of total RNA in a DNA Mouse Gene ST 1.0 microarray (Affymetrix) at the Genomic Technologies Unit (Biological Services, WIS). Analysis was conducted at the Bioinformatics Unit (Biological Services, WIS). Microarray analysis was carried out using Partek Genomic Suite (Inc. St. Charles, MO; www.partek. com). The raw probe intensities were adjusted based on the number of G and C bases in the probe sequence, before any probe correction. Preprocessing was performed using the Robust Microarray Averaging algorithm [34]. The normalized data were explored by principal component analysis and hierarchical clustering to detect batch or other random effects that may appear if the replicates are carried out sequentially. Batch effects were corrected using the defaults of the software. Genes expressed below background levels in all arrays were removed from further analyses. Analysis of variance (ANOVA) including contrasts was applied to the data set. P-values for gene enrichment were calculated using the hypergeometric distribution. Gene list for TLR pathway was taken from Kegg. Gene list for osteogenesis was taken from the list of genes on SAbioscince’s osteogenesis mouse PCR array. Microarray data are available at the Gene Expression Omnibus (GEO) website, accession number GSE50560 at the following link: http://www.ncbi.nlm.nih. gov/geo/query/acc.cgi?token=fpqrdoioucqiojk&acc= GSE50560. IL-6 Induction by rLBP MSCs at 7×103 cells/well were seeded in MSC medium in 96well plates. Later (24 h), the medium was replaced with DMEM containing rLBP or CCL5 (R&D Systems, Minneapolis, MN) and LPS, Pam3Cys or CpG. After 72 h, conditioned medium was analyzed for IL-6 detection by ELISA. LBP Neutralizing Assay MSCs at 1×104 cells/well were seeded in MSC medium in 96well plates. Later (24 h), the medium was replaced with DMEM containing 30 μg/ml isotype control (BioLegend) or anti-LBP neutralizing antibody (Clone biG 33, EnzoLife Sciences, Lörrach, Germany). 0.1 μg/ml of Pam3Cys or LPS was added to the cultures. After 4 h, conditioned medium was analyzed for IL-6 detection in ELISA. Regulation of Osteogenic Differentiation by TGFβ1 MSCs at 6×104 or 9×104cells/well were seeded in MSC medium in 96-well plates. Later (24 h), the medium was replaced with osteogenic induction medium, with or without 0.5 ng/ml IL-1β and TGFβ1. Medium was replaced twice a
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week. After 10 days, cultures were fixed and stained with alizarin red. Inhibition of TGFβ Receptor during Osteogenic Differentiation MSCs at 6×104 cells/well were seeded in MSC medium in 96well plate. Later (24 h), the medium was replaced with osteogenic induction medium, containing vehicle (DMSO) or TGFβ receptor inhibitor SB431542 (InvivoGen, San Diego, California) at the noted concentrations, with or without IL-1β. Medium was replaced twice a week. After 10 days, cultures were fixed and stained with alizarin red. Statistical Analysis The JMP5 program was used for statistical analysis by using the Kruskal-Wallis one-way analysis of variance, 2-sided and the GraphPad Instat 3 program was used for ANOVA. Differences were considered statistically significant with a P value less than 0.05.
Results TLR Ligands Trigger Divergent Differentiation Responses in MSC Populations Independently derived cultured mesenchymal cells are a priori heterogeneous in their differentiation potencies; whereas some preparations are multipotent, other isolates exhibit oligopotency or, at the other extreme, do not have any detectable differentiation capacity at all [17]. This is often attributed to a hierarchy in the MSC differentiation cascade, initiating from a multipotent stem cell that gives rise to oligopotent progenitor and ending with a fully differentiated progeny (reviewed in [17]). To eliminate this type of heterogeneity from our experimental system, we selected tri-potent MSCs, capable at least of osteogenic, chondrogenic and adipogenic differentiation. Five such independent populations, marked I to V, were isolated and propagated (Figure 1S A). The selected MSCs expressed Sca-I and lacked expression of hematopoietic cell surface markers, with the exception of CD34 (Figure 1S B). All MSCs expressed TLR1-9 mRNA at different titers (Figure 1S C). Ligands for several TLRs, including TLR2/1—Pam3Cys; TLR2/6—PG; TLR3—Poly(I:C) and TLR4—LPS, have been previously shown to induce IL-6 secretion by MSCs [33], and were tested here. We used this property here as a means to analyze the extent of heterogeneity among the five independent MSC isolates. Basal secretion of IL-6 was observed in MSC IV alone. All MSC populations secreted significantly higher levels of IL-6 in response to the TLR2 ligands Pam3Cys and PG, varying between 7.8
−65 ng/ml and 3.8–69.3 ng/ml respectively. LPS induced IL-6 secretion in all MSCs with the exception of isolates II and IV, varying between 7.1–42 ng/ml. Poly(I:C) induced IL6 secretion in MSCs I, II and V varying between 30 pg/ml– 11.1 ng/ml (Fig. 1a). Thus, the TLRs expressed by MSCs appear to be functionally active and their activation elicited an IL-6 secretion response in some but not all cases, which varied in its magnitude among the different MSC isolates. Further experiments were then aimed at determining the effect of TLR ligands on the differentiation capacity of MSCs. A previous study showed that Pam3Cys inhibits the differentiation of a MSC isolate (MSC I studied here) towards the osteogenic, adipogenic and chondrogenic pathways [33]. This finding was confirmed herein, as Pam3Cys inhibited osteogenic differentiation of the previously studied MSC I (Fig. 1b and c). However, this response was not common to all MSC preparations, e.g., the differentiation of MSC III and IV was unaltered by Pam3Cys, whereas this ligand did modify the differentiation of other isolates. Another example for such heterogeneity can be observed for TLR3 ligand Poly(I:C), which had no effect on MSCs I, III and IV, augmented the differentiation of MSC II and inhibited the differentiation of MSC V. It is therefore concluded that independent MSC isolates diverge from one another to the extent that they even show opposite osteogenic differentiation responses to the same TLR ligand. It is noteworthy that osteogenic differentiation of early passaged MSC populations (P4-P6) was highly divergent in response to TLR activation and in some occasions even changed during passaging (Figure 2S). Thus, the different and often opposing responses of independent MSC isolates to the same ligand were not due to prolonged in vitro propagation. MSC proliferation and migration have been previously shown to be affected by TLR activation [33]. While MSC differentiation is shown herein to be modified in a divergent manner, the response of MSCs to TLR activation in terms of proliferation and migration was mostly uniform: Pam3Cys inhibited MSC migration of all isolates, and augmented the proliferation of 4 out of 5 MSC (Figure 3S). Overall, the analysis of consequences of TLR activation on a group of independently isolated MSCs lead to the conclusion that these cell populations markedly differ in their responses in terms of differentiation and IL-6 secretion. The proliferation and migration of these cells were affected by TLR activation but the trend in all isolates was essentially similar. MSC Clones from the Bone Marrow of an Individual Mouse Exhibit Inherent Heterogeneity MSCs are derived by culturing a bulk of bone marrow cells, containing a large number of colony forming units-fibroblasts (CFU-Fs). This must result in a polyclonal population. To
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Fig. 1 MSCs I-V respond differently to TLR activation. a MSCs I-V were treated with Pam3Cys, PG, LPS or Poly(I:C) for 24 h. Conditioned media were collected and assayed by ELISA for the presence of IL-6. Representative experiment out of at least 3 repeats is presented. b MSCs were induced to differentiate into osteocytes with or without 20 μg/ml Pam3Cys, PG, LPS or Poly(I:C). After up to 3 weeks, cell cultures were fixed and stained with Alizarin red. Original magnifications: ×10.
Representative pictures from at least 4 repeats are presented. Summary of osteogenic differentiation in the presence of TLR ligands: No change ), augmentation ( ) or inhibition ( ). c Alizarin Red stain was ( extracted from MSC I-V and quantified. ((*) p
Divergent Levels of LBP and TGFβ1 in Murine MSCs Lead to Heterogenic Response to TLR and Proinflammatory Cytokine Activation Sarit Levin & Meirav Pevsner-Fischer & Sivan Kagan & Hila Lifshitz & Ada Weinstock & Diana Gataulin & Gilgi Friedlander & Dov Zipori
# Springer Science+Business Media New York 2014
Abstract The outstanding heterogeneity of stem cell populations is a major obstacle on the way to their clinical application. It is therefore paramount to identify the molecular mechanisms that underlay this heterogeneity. Individually derived bone marrow mesenchymal stromal cells (MSCs) preparations, studied here, diverged markedly in various properties, despite of being all tripotent in their differentiation potential. Microarray analysis showed that MSC diversity is evident also in highly variable gene expression patterns. Differentially expressed genes were significantly enriched in toll-like receptors (TLRs) and differentiation pathways. Marked differences were observed in LPS binding protein (LBP) and transforming growth factor (TGF)β1 expression. These differences correlated with MSC functionality. Therefore, the possible contribution of these molecules to MSC diversity was examined. In the TLR signaling pathway, LBP levels predicted the ability of specific MSCs to secrete interleukin (IL)-6 in response to LPS. A relatively higher expression of TGFβ1 endowed MSCs with a capacity to respond to IL-1β by reduced Sarit Levin and Meirav Pevsner-Fischer contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s12015-014-9498-z) contains supplementary material, which is available to authorized users. S. Levin : M. Pevsner-Fischer : S. Kagan : H. Lifshitz : A. Weinstock : D. Gataulin : D. Zipori Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel 76100 G. Friedlander The Biological Services Unit, Weizmann Institute of Science, Rehovot, Israel 76100 D. Zipori (*) Department of Molecular Cell Biology, Weizmann Institute of Science, 234 Herzel St., Rehovot 7632700, Israel e-mail: [email protected] URL: http://www.weizmann.ac.il/mcb/Zipori/
osteogenic differentiation. This study thus demonstrates major diversity within MSC isolates, which appears early on following derivation and persists following long–term culture. MSC heterogeneity results from highly variable transcriptome. Differential expression of LBP and TGFβ1, along with other genes, in different MSC preparations, produces the variable responses to external stimuli. Keywords MSC . Heterogeneity . LBP . TGFβ1 . TLR
Introduction Mesenchymal stromal cells (MSCs) are adult progenitors that were originally obtained by expansion of the bone marrow (BM) plastic-adherent cell fraction [1, 2]. Their capacity to differentiate in vitro into multiple mesodermal lineages is used as a functional, retrospective defining criterion [1–3]. MSCs display a variety of functions, including the support and regulation of hematopoiesis in vivo and in vitro[4–6], regulation of immune responses [7–11], migration into injured tissues and participation in the repair of tissue damage [12–16]. The heterogeneity of BM-MSCs is a well-known phenomenon. Individual MSC preparations differ in morphology and in their growth rate and differentiation potentials [17]. A hierarchical model was proposed to explain the existence of MSC clones with divergent differentiation potential. Multi-potent mouse and rat MSCs were suggested to give rise to more restricted clones and to finally differentiate into mono-potent progenitor cells [18]. This process was suggested to be a nonrandom, single-step process in which multi-potential progenitors become exclusively restricted to a single lineage by particular culture conditions and inducers [19–21], but this view is still a matter of debate [22–24].
Stem Cell Rev and Rep
We and others have previously showed that cultured murine MSCs express toll-like receptors (TLR)s [25–32]. The activation of MSCs by TLR ligands induced interleukin-6 (IL6) secretion and NFкB nuclear translocation. Pam3Cys, a prototypic ligand for TLR2, induced proliferation of MSCs and regulated their differentiation [33]. It was also shown that TLR activation regulates human MSC differentiation, proliferation, immune regulation and migration [25–32]. However, there is a lack of consensus in relation to the mode by which MSCs respond to TLR ligands due to conflicting reports on various effects of TLR activation on MSCs. The present study aims to investigate the molecular basis for the heterogeneity in the response of MSCs to TLR activation and its origin. To exclude differences resulting form the position of the cells within the hierarchical lineage, we chose to examine tripotent MSCs only. It is shown herein that independent isolates of MSCs differ markedly in their functional properties. Transcriptome analysis of such divergent populations provides means to discover new regulatory pathways.
Materials and Methods Mice BALB/C and C57BL/6J mice were purchased from Harlan Olac. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of the Weizmann Institute of Science. Permit Number: 04400810-1. Cell Culture MSCs were grown in murine MesenCult Basal Media supplemented with 20 % murine mesenchymal supplement (StemCell Technologies, Vancouver, Canada), 60 μg/mL penicillin, 100 μg/mL streptomycin and 50 μg/mL kanamycin. MSCs from passages 4–6 and 14 to passage 20 were used in all experiments described. Reagents Escherichia coli O55:B5 LPS, peptidoglycan of Staphylococcus aureus, and polyinosinicpolycytidylic acid (Poly(I:C)) were purchased from Sigma (Rehovot, Israel). Pam3Cys was purchased from EMC microcollections (Tübingen, Germany). CpG—the phosphorothioaete oligonucleotides—was synthesized at the Oligonucleotide Synthesis Unit of the Weizmann Institute of Science (Rehovot, Israel). The oligonucleotide CpG contains 2 9-mer segments: 5′-TCCATAACGTTGCA AACGTTCTG-3′. TNFα, IL-1β and human TGFβ1 were
purchased from PeproTech/Cytolab (Rehovot, Israel). Polyclonal rabbit anti-ERK1/2, anti-JNK and anti-p38 were purchased from Sigma (Rehovot, Israel). Polyclonal rabbit anti– NFкB p65 was obtained from eBioscience (San Diego, CA). Polyclonal rabbit anti-Histone3 was obtained from Open biosystems (Huntsville, AL). Anti-phospho JNK and antiphospho ERK1/2 were purchased from Santa-Cruz Biotech (Santa Cruz, CA). Anti-phospho p38 was purchased from BioSource Invitrogen (Carlsbad, CA). BM Cell Extraction and MSC Production BM cells were obtained from femur and tibia of 6- to 8-weekold C57BL/6J mice, re-suspended in PBS with 1 % FBS and subjected to centrifugation. The cells were then seeded in 60mm plates containing MSC medium. Half of the medium was replaced every 3 days; upon confluence, the cells were removed using trypsin (0.05 % EDTA, 0.25 % trypsin; Biological Industries) and reseeded. For clone isolation, fresh BM cells from femur and tibia of 6–8 week-old BALB/C mice were seeded in 24-well plates (Falcon) at a concentration of 65 or 30×105 cells per well and propagated in MSC medium. Only wells containing single colonies were used for further experiments. Cell Sorting Cell sorting was conducted for MSC I as described in our previous work [33]. Evaluation of MSC Differentiation Osteogenesis Cells were seeded at a concentration of 3× 104cells/well in a 24-well plate. The next day, osteogenic medium containing 50 μg/mL L-ascorbic acid-2 phosphate, 10 mM glycerol 2-phosphate disodium salt, and 1×10−7 M dexamethasone (all from Sigma) were added, either with or without pam3cys, peptidoglycan, LPS, Poly(I:C), TNFα or IL-1β in the indicated concentrations. The cells were grown for 1 to 3 weeks with medium replacement twice a week. Osteogenic differentiation was detected by alizarin red staining. For alizarin red quantification, 0.5 N HCl and 5 % sodium dodecyl sulfate (SDS) were added to each well. Light absorbance by the extracted dye was measured in 405 nm. Real time-PCR Total RNA was isolated from confluent MSCs using TRI reagent (MRC, Cincinnati, OH). Contaminating DNA was removed using DNase treatment by TURBO DNA-free™ Kit (Ambion). First-strand cDNA synthesis was performed from 5 μg of total RNA by extension of oligo(dT) primers with 200 U Moloney murine leukemia virus reverse
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transcriptase (MMLV-RT; Promega, Madison, WI). Real-time PCR was performed on an ABI 7000 real-time PCR system, using SYBR green PCR master mix (Applied Biosystems, Lincoln Center, CA). The values for the specific genes were normalized to HPRT. Primer sequences used: LBP F: 5′TTTCATAGCCTGGAGATTCAGAACT-3′. LBP R: 5′TGGCCTGGGAGCAGCTT-3′. TGFβ1 F: 5′- TGAACC AAGGAGACGGAATACA-3′. TGFβ1 R: 5′- AAGAGCAG TGAGCGCTGAATC-3′. HPRT F: 5′- GCAGTACAGCCC CAAAATGG-3′. HPRT R: 5′- GGTCCTTTTCACCAGCAA GCT-3′. IL-6 ELISA MSCs at 1.8×104 cells/well were seeded in MSC medium in 96-well plates. Later (24 h), the medium was replaced with DMEM (Gibco/BRL, Gaithersburg, MD) and supplemented with 10 % FBS with or without all TLR ligands in the indicated concentrations. IL-6 concentration in culture media was determined by enzyme-linked immunosorbent assay (ELISA) for IL-6 (OptiEIA kit; BD Pharmingen, San Diego, CA) according to the manufacturer’s instructions. Standard curves were established using mouse recombinant IL-6. Western Blot Analysis For NF-kB detection, cell lysates were microcentrifuged at 950×g for separation of nuclear (pellet) and cytoplasmic (supernatant) fraction. The nuclear fraction was then lysed in a buffer containing 30 mM HEPES, 450 mM NaCl, 25 % glycerol, 0.5 mM EDTA, 12 mM MgCl2, 6 mM DTT, 1 mM PMSF, and protease and phosphatase inhibitors. Equal amounts of protein were loaded and electrophoresed on a 12 % SDS–polyacrylamide gel electrophoresis (SDSPAGE), transferred to nitrocellulose membranes, blocked and treated overnight with a rabbit polyclonal anti-Histone 3, rabbit polyclonal anti–ERK1/2, or rabbit polyclonal anti– NF-kB in PBS containing 0.05 % tween (PBST) with 1 % BSA. Following washing, the membranes were incubated with horseradish peroxidase (HRP)–conjugated anti–rabbit IgG antibody in PBST with 2.5 % skimmed milk. Electrochemiluminescence (ECL) detected the immunoreactive protein. For ERK1/2, JNK or P38 phosphorylation detection, equal amounts of whole cell lysates were loaded and electrophoresed on a 12 % SDS-PAGE, transferred to nitrocellulose membranes, blocked and treated overnight with either a anti-ERK1/2, anti-JNK, anti-p38, anti-phospho p38, anti-phospho JNK or anti-phospho ERK1/2 in PBS PBST with 1 % BSA. Following washing, the membranes were incubated with HRP–conjugated anti–rabbit or mouse IgG antibody in PBST with 2.5 % skimmed milk. ECL detected the immunoreactive protein.
Microarray Analysis Experiment was carried out using 100 ng of total RNA in a DNA Mouse Gene ST 1.0 microarray (Affymetrix) at the Genomic Technologies Unit (Biological Services, WIS). Analysis was conducted at the Bioinformatics Unit (Biological Services, WIS). Microarray analysis was carried out using Partek Genomic Suite (Inc. St. Charles, MO; www.partek. com). The raw probe intensities were adjusted based on the number of G and C bases in the probe sequence, before any probe correction. Preprocessing was performed using the Robust Microarray Averaging algorithm [34]. The normalized data were explored by principal component analysis and hierarchical clustering to detect batch or other random effects that may appear if the replicates are carried out sequentially. Batch effects were corrected using the defaults of the software. Genes expressed below background levels in all arrays were removed from further analyses. Analysis of variance (ANOVA) including contrasts was applied to the data set. P-values for gene enrichment were calculated using the hypergeometric distribution. Gene list for TLR pathway was taken from Kegg. Gene list for osteogenesis was taken from the list of genes on SAbioscince’s osteogenesis mouse PCR array. Microarray data are available at the Gene Expression Omnibus (GEO) website, accession number GSE50560 at the following link: http://www.ncbi.nlm.nih. gov/geo/query/acc.cgi?token=fpqrdoioucqiojk&acc= GSE50560. IL-6 Induction by rLBP MSCs at 7×103 cells/well were seeded in MSC medium in 96well plates. Later (24 h), the medium was replaced with DMEM containing rLBP or CCL5 (R&D Systems, Minneapolis, MN) and LPS, Pam3Cys or CpG. After 72 h, conditioned medium was analyzed for IL-6 detection by ELISA. LBP Neutralizing Assay MSCs at 1×104 cells/well were seeded in MSC medium in 96well plates. Later (24 h), the medium was replaced with DMEM containing 30 μg/ml isotype control (BioLegend) or anti-LBP neutralizing antibody (Clone biG 33, EnzoLife Sciences, Lörrach, Germany). 0.1 μg/ml of Pam3Cys or LPS was added to the cultures. After 4 h, conditioned medium was analyzed for IL-6 detection in ELISA. Regulation of Osteogenic Differentiation by TGFβ1 MSCs at 6×104 or 9×104cells/well were seeded in MSC medium in 96-well plates. Later (24 h), the medium was replaced with osteogenic induction medium, with or without 0.5 ng/ml IL-1β and TGFβ1. Medium was replaced twice a
Stem Cell Rev and Rep
week. After 10 days, cultures were fixed and stained with alizarin red. Inhibition of TGFβ Receptor during Osteogenic Differentiation MSCs at 6×104 cells/well were seeded in MSC medium in 96well plate. Later (24 h), the medium was replaced with osteogenic induction medium, containing vehicle (DMSO) or TGFβ receptor inhibitor SB431542 (InvivoGen, San Diego, California) at the noted concentrations, with or without IL-1β. Medium was replaced twice a week. After 10 days, cultures were fixed and stained with alizarin red. Statistical Analysis The JMP5 program was used for statistical analysis by using the Kruskal-Wallis one-way analysis of variance, 2-sided and the GraphPad Instat 3 program was used for ANOVA. Differences were considered statistically significant with a P value less than 0.05.
Results TLR Ligands Trigger Divergent Differentiation Responses in MSC Populations Independently derived cultured mesenchymal cells are a priori heterogeneous in their differentiation potencies; whereas some preparations are multipotent, other isolates exhibit oligopotency or, at the other extreme, do not have any detectable differentiation capacity at all [17]. This is often attributed to a hierarchy in the MSC differentiation cascade, initiating from a multipotent stem cell that gives rise to oligopotent progenitor and ending with a fully differentiated progeny (reviewed in [17]). To eliminate this type of heterogeneity from our experimental system, we selected tri-potent MSCs, capable at least of osteogenic, chondrogenic and adipogenic differentiation. Five such independent populations, marked I to V, were isolated and propagated (Figure 1S A). The selected MSCs expressed Sca-I and lacked expression of hematopoietic cell surface markers, with the exception of CD34 (Figure 1S B). All MSCs expressed TLR1-9 mRNA at different titers (Figure 1S C). Ligands for several TLRs, including TLR2/1—Pam3Cys; TLR2/6—PG; TLR3—Poly(I:C) and TLR4—LPS, have been previously shown to induce IL-6 secretion by MSCs [33], and were tested here. We used this property here as a means to analyze the extent of heterogeneity among the five independent MSC isolates. Basal secretion of IL-6 was observed in MSC IV alone. All MSC populations secreted significantly higher levels of IL-6 in response to the TLR2 ligands Pam3Cys and PG, varying between 7.8
−65 ng/ml and 3.8–69.3 ng/ml respectively. LPS induced IL-6 secretion in all MSCs with the exception of isolates II and IV, varying between 7.1–42 ng/ml. Poly(I:C) induced IL6 secretion in MSCs I, II and V varying between 30 pg/ml– 11.1 ng/ml (Fig. 1a). Thus, the TLRs expressed by MSCs appear to be functionally active and their activation elicited an IL-6 secretion response in some but not all cases, which varied in its magnitude among the different MSC isolates. Further experiments were then aimed at determining the effect of TLR ligands on the differentiation capacity of MSCs. A previous study showed that Pam3Cys inhibits the differentiation of a MSC isolate (MSC I studied here) towards the osteogenic, adipogenic and chondrogenic pathways [33]. This finding was confirmed herein, as Pam3Cys inhibited osteogenic differentiation of the previously studied MSC I (Fig. 1b and c). However, this response was not common to all MSC preparations, e.g., the differentiation of MSC III and IV was unaltered by Pam3Cys, whereas this ligand did modify the differentiation of other isolates. Another example for such heterogeneity can be observed for TLR3 ligand Poly(I:C), which had no effect on MSCs I, III and IV, augmented the differentiation of MSC II and inhibited the differentiation of MSC V. It is therefore concluded that independent MSC isolates diverge from one another to the extent that they even show opposite osteogenic differentiation responses to the same TLR ligand. It is noteworthy that osteogenic differentiation of early passaged MSC populations (P4-P6) was highly divergent in response to TLR activation and in some occasions even changed during passaging (Figure 2S). Thus, the different and often opposing responses of independent MSC isolates to the same ligand were not due to prolonged in vitro propagation. MSC proliferation and migration have been previously shown to be affected by TLR activation [33]. While MSC differentiation is shown herein to be modified in a divergent manner, the response of MSCs to TLR activation in terms of proliferation and migration was mostly uniform: Pam3Cys inhibited MSC migration of all isolates, and augmented the proliferation of 4 out of 5 MSC (Figure 3S). Overall, the analysis of consequences of TLR activation on a group of independently isolated MSCs lead to the conclusion that these cell populations markedly differ in their responses in terms of differentiation and IL-6 secretion. The proliferation and migration of these cells were affected by TLR activation but the trend in all isolates was essentially similar. MSC Clones from the Bone Marrow of an Individual Mouse Exhibit Inherent Heterogeneity MSCs are derived by culturing a bulk of bone marrow cells, containing a large number of colony forming units-fibroblasts (CFU-Fs). This must result in a polyclonal population. To
Stem Cell Rev and Rep
Fig. 1 MSCs I-V respond differently to TLR activation. a MSCs I-V were treated with Pam3Cys, PG, LPS or Poly(I:C) for 24 h. Conditioned media were collected and assayed by ELISA for the presence of IL-6. Representative experiment out of at least 3 repeats is presented. b MSCs were induced to differentiate into osteocytes with or without 20 μg/ml Pam3Cys, PG, LPS or Poly(I:C). After up to 3 weeks, cell cultures were fixed and stained with Alizarin red. Original magnifications: ×10.
Representative pictures from at least 4 repeats are presented. Summary of osteogenic differentiation in the presence of TLR ligands: No change ), augmentation ( ) or inhibition ( ). c Alizarin Red stain was ( extracted from MSC I-V and quantified. ((*) p
