Stem Cells http://www.stemcellsportal.com/
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

First published online March 29, 2007
Stem Cells Vol. 25 No. 7 July 2007, pp. 1737 -1745
doi:10.1634/stemcells.2007-0054; www.StemCells.com
© 2007 AlphaMed Press

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2007-0054v1
25/7/1737    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ponte, A. L.
Right arrow Articles by Domenech, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ponte, A. L.
Right arrow Articles by Domenech, J.

TISSUE-SPECIFIC STEM CELLS

The In Vitro Migration Capacity of Human Bone Marrow Mesenchymal Stem Cells: Comparison of Chemokine and Growth Factor Chemotactic Activities

Adriana López Pontea, Emeline Maraisa, Nathalie Gallaya, Alain Langonnéa,b, Bruno Delormea, Olivier Héraulta,c, Pierre Charborda, Jorge Domenecha,c

aLaboratoire d'Hématopoïèse, Institut National de la Santé et de la Recherche Médicale ERI5, IFR135 Université François Rabelais, Tours, France;
bEFS Centre-Atlantique, Tours, France;
cCHRU de Tours, Tours, France

Key Words. Bone marrow • Mesenchymal stem cell • In vitro migration • Growth factors • Chemokines • Tumor necrosis factor {alpha}

Correspondence: Jorge Domenech M.D., Ph.D., Laboratoire d'Hématopoïèse, Inserm-ESPRI-EA3855, Faculté de Médecine, 10 boulevard Tonnellé, 37 032 Tours Cedex, France. Telephone: 33-247-47-47-21; Fax: 33-247-47-69-34; e-mail: domenech{at}med.univ-tours.fr

Received January 24, 2007; accepted for publication March 20, 2007.
First published online in STEM CELLS EXPRESS   March 29, 2007.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures of Potential...
 Acknowledgments
 References
 
Adult bone marrow (BM)-derived stem cells, including hematopoietic stem cells (HSCs) and MSCs, represent an important source of cells for the repair of a number of damaged tissues. In contrast to HSCs, the soluble factors able to induce MSC migration have not been extensively studied. In the present work, we compared the in vitro migration capacity of human BM-derived MSCs, preincubated or not with the inflammatory cytokines interleukin 1ß (IL1ß) and tumor necrosis factor {alpha} (TNF{alpha}), in response to 16 growth factors (GFs) and chemokines. We show that BM MSCs migrate in response to many chemotactic factors. The GFs platelet-derived growth factor-AB (PDGF-AB) and insulin-like growth factor 1 (IGF-1) are the most potent, whereas the chemokines RANTES, macrophage-derived chemokine (MDC), and stromal-derived factor-1 (SDF-1) have limited effect. Remarkably, preincubation with TNF{alpha} leads to increased MSC migration toward chemokines, whereas migration toward most GFs is unchanged. Consistent with these results, BM MSCs express the tyrosine kinase receptors PDGF-receptor (R) {alpha}, PDGF-Rß, and IGF-R, as well as the RANTES and MDC receptors CCR2, CCR3, and CCR4 and the SDF-1 receptor CXCR4. TNF{alpha} increases CCR2, CCR3, and CCR4 expression (as opposed to that of CXCR4), together with RANTES membrane binding. These data indicate that the migration capacity of BM MSCs is under the control of a large range of receptor tyrosine kinase GFs and CC and CXC chemokines. Most chemokines are more effective on TNF{alpha}-primed cells. Our results suggest that the mobilization of MSCs and their subsequent homing to injured tissues may depend on the systemic and local inflammatory state.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures of Potential...
 Acknowledgments
 References
 
Adult bone marrow (BM)-derived stem cells represent an important source of cells for repair of a number of damaged tissues. As shown by studies in vitro [1, 2] and in vivo [3, 4], these cells include two distinct multipotential cell populations: hematopoietic stem cells (HSCs), which differentiate into all hematopoietic lineages, and MSCs, which give rise to the different hematopoietic microenvironmental cells, including vascular smooth muscle-like stromal cells, adipocytes, osteoblasts, and, more controversially, endothelial cells. MSCs also generate cells that do not belong to the hematopoietic microenvironment that are mesodermal in origin, such as chondrocytes [3] and cardiac or skeletal muscle cells [5, 6], but may also be neuroectodermal (neurons, astrocytes, oligodendrocytes) [7, 8] or endodermal (hepatocytes) [9, 10].

BM-derived stem cells have been currently used in humans for more than 30 years by transplanting HSCs for hematopoietic rescue after high-dose therapy [11]. By contrast, there are fewer reports on the clinical use of MSCs in patients. Preclinical models with infusion of MSCs in animals have provided evidence for their local engraftment and/or repair capacity in settings such as cerebral injury [12], myocardial ischemia/infarction [6], muscular dystrophy [5], bone disease [13], and bone fractures [14]. In addition, coadministration of human marrow MSCs and HSCs to preimmune fetal sheep has been shown to improve the hematopoietic reconstitution [15], and this finding has been confirmed after transplantation in humans [16]. Another recent application of MSC cotransplantation with allogenic HSCs is the cure of severe graft-versus-host disease [17].

As reported in murine [18], simian [19–21], and human [22] models, following systemic infusion into the bloodstream, BM MSCs are able to colonize and persist long-term in a wide range of tissues, including, in addition to BM, muscle, skin, gut, liver, and lung. Otherwise, MSCs can be induced to circulate into peripheral blood in animal models under particular conditions, such as hypoxia [23]. The chemotactic signals that guide MSCs to appropriate microenvironments or induce their circulation are yet to be clarified, which is not the case for HSCs, for which the predominant role of the chemokine SDF-1 and its receptor CXCR-4 is now well-established [24]. Some soluble factors have recently been reported to exert chemotactic effects on BM MSCs, including chemokines [25–27] and growth factors (GFs) [28–30], but their respective physiological relevance remains unclear. A better knowledge of the chemotactic factors effective on MSCs would be of clinical interest since modulation of their activity could affect not only the engraftment efficiency to damaged sites but also the mobilization into PB. In the present work, we studied the in vitro migration capacity of human BM-derived MSCs in response to 16 chemokines and GFs. We show that, in contrast to what is known for HSCs, a wide range of soluble factors exert significant chemotactic activity on MSCs and that some GFs are better chemoattractants than chemokines. Remarkably, inflammatory cytokines, such as tumor necrosis factor {alpha} (TNF{alpha}), are able to increase the sensitivity of MSCs to chemokines.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures of Potential...
 Acknowledgments
 References
 
Cell Cultures
BM MSCs were obtained from informed and consenting patients undergoing orthopedic surgery (Orthopedic Surgery Department, Trousseau Hospital, Tours, France), following a procedure approved by the local ethical committee. Cells harvested from the iliac crest were centrifuged, and nucleated cells were counted and seeded at density of 5 x 104 cells per cm2 in {alpha}-minimum essential medium (Invitrogen, Cergy-Pontoise, France, http://www.invitrogen.com) supplemented with 10% (vol/vol) screened fetal calf serum (FCS), 20 µmol/l L-glutamine (Invitrogen), and 100 units/ml penicillin G (Invitrogen). Cells were incubated at 37°C in 95% humidified air and 5% CO2. On day 2, all nonadherent cells were removed by changing the medium; thereafter, medium was changed twice a week. When fully confluent, the layer of adherent cells was trypsinized (0.25% trypsin-EDTA, Invitrogen), and the cells were resuspended in culture medium and seeded at 1 x 103 cells per cm2 (passage 1 [P1]). BM MSCs were used at P2 in all experiments (migration assays, flow cytometry analyses, and real time (RQ)-polymerase chain reaction [PCR]).

The three-lineage mesenchymal differentiation of the BM MSCs used was systematically checked by culturing cells in adipogenic, chondrogenic, and osteogenic induction media as previously described [31], and the lack of remaining hematopoietic CD45+ cells was verified by flow cytometry analysis.

Cytokine Prestimulation of BM MSCs
Confluent P2 BM MSCs were preincubated for 1 day with fresh culture medium alone or supplemented with 1 ng/ml TNF{alpha} (R&D Systems, Lille, France, http://www.rndsystems.com) or interleukin 1ß (IL1ß) (R&D Systems) and then trypsinized for flow cytometry analyses and RQ-PCR. For migration assays, confluent P2 BM MSCs were preincubated with migration medium consisting of RPMI medium with 0.25% bovine serum albumin (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) (instead of culture medium) supplemented with 1 ng/ml TNF{alpha} or IL1ß.

Migration Assays
Migration assays were performed in transwell dishes (Corning Costar, Cambridge, MA, http://www.corning.com/lifesciences) 6.5 mm in diameter with 8-µm pore filters. The upper side of the transwell filter was coated for 1 hour at 37°C with 0.1% (wt/vol) bovine gelatin (Sigma-Aldrich) in phosphate-buffered saline (PBS). Unstimulated or stimulated P2 BM MSCs (5 x 105 cells) were added to the upper chamber, and 600 µl of migration medium with chemotactic factors was added to the bottom chamber. Migration observed in the presence of 30% FCS and with medium alone served as positive and negative controls, respectively. After overnight incubation of the transwells at 37°C, 5% CO2, the upper side of the filters was carefully washed with cold PBS, and cells remaining on the upper face of the filters were removed with a cotton wool swab. Transwell filters were stained using May-Grünwald-Giemsa, cut out with a scalpel, and mounted onto glass slides, putting the lower face on the top. The total number of cells that had migrated were counted using light microscopy at x200 magnification. Each experiment was performed in duplicate. Data were expressed as numbers of total migrated cells per insert or as percentages of cells related to that of the negative control. We evaluated the chemotactic activity of CC chemokines (100 ng/ml MCP-1, 150 ng/ml RANTES, 50 ng/ml MIP-1{alpha}, 100 ng/ml Eotaxin-1, 100 ng/ml Eotaxin-2, 100 ng/ml macrophage-derived chemokine [MDC]), CXC chemokines (50 ng/ml GRO-{alpha}, 150 ng/ml SDF-1), CX3C chemokine (300 ng/ml Fractalkine), and growth factors (10 ng/ml fibroblast growth factor-2 [FGF-2], 10 ng/ml platelet-derived growth factor-AB [PDGF-AB], 50 ng/ml hepatocyte growth factor [HGF], 10 ng/ml epidermal growth factor [EGF], 10 ng/ml vascular endothelial growth factor [VEGF], 30 ng/ml insulin-like growth factor-1 [IGF-1], 10 ng/ml Angiopoietin-1 [Ang-1]). All cytokines used were recombinant factors of human origin (rh), purchased from PeproTech (Rocky Hill, NJ, http://www.peprotech.com) or from R&D Systems. Concentrations of GFs and chemokines used were chosen that showed optimal promigratory activity according to the literature.

Flow Cytometry Analysis
Unstimulated and TNF{alpha}-stimulated BM MCSs were analyzed for membrane receptor expression using a three-step labeling procedure. Cells were incubated at 4°C for 45 minutes with the following primary mouse anti-human monoclonal antibodies (McAbs) to growth factor and chemokine receptors: anti-PDGF receptor (R) {alpha} (clone PRa292), anti-PDGF-Rß (clone PR7212), anti-EGF-R (clone 102618), anti-FGF-R2 (clone 98725), anti-HGF-R (clone D0-24), anti-TIE-2 (clone 83715), anti-CCR1 (clone 53504), anti-CCR2 (clone 48607), anti-CCR3 (clone 61828), anti-CCR4 (clone 205410), anti-CCR5 (clone 45502.111), anti-CXCR1 (clone 5A12), anti-CXCR2 (clone 6C6), anti-CXCR4 (clone 44717), anti-CXCR5 (clone 51505), and anti-CX3CR1 (clone 2A9-1). All McAbs were purchased from R&D Systems, except for anti-HGF-R, from Upstate (Billerica, MA, http://www.upstate.com), anti-CXCR1 and anti-CXCR2, from Becton, Dickinson and Company (Le-Pont-de-Claix, France, http://www.bd.com), and anti-CX3CR1, from MBL (Nagoya, Japan, http://www.mblintl.com). Cells were subsequently incubated at 4°C with biotinylated goat anti-mouse Ig antibody (Ab) (Becton Dickinson) for 1 hour and with R-phycoerythrin-conjugated streptavidin (Dako, Trappes, France, http://www.dako.com) for 45 minutes. As a negative control, cells were incubated with irrelevant mouse IgG1, IgG2a, or IgG2b McAbs (Becton Dickinson). A minimum of 20,000 events was recorded for each analysis, using a FACSCalibur (Becton Dickinson) flow cytometer. To exclude dead cells, cells were stained before analysis with 7-aminoactinomycin D (7-AAD) in PBS. Appropriate gating was performed using the forward scatter versus side scatter dot plot and gating out 7-AAD-positive dead cells. Expression of membrane receptors was studied on a FL2-H logarithmic scale. For each antibody, the histogram was compared with that of the irrelevant negative control.

Binding Assays with Labeled Cytokines
The binding of RANTES and stromal-derived factor (SDF)-1{alpha} to unstimulated and TNF{alpha}-stimulated BM MSCs was measured with the Fluorokine kit (R&D Systems). Cells were incubated with a biotinylated rhRANTES or rhSDF-1{alpha} at 4°C for 60 minutes. Stained cells were pretreated with purified mouse Ig (10 µg per 106 cells) for 15 minutes at room temperature to block Fc-mediated interactions. Cells were then incubated (without washing) with avidin-fluorescein isothiocyanate reagent for 30 minutes. All samples were labeled with 7-AAD before analysis with a FACSCalibur (Becton Dickinson) flow cytometer. In parallel experiments, for specificity testing, polyclonal goat IgG anti-human RANTES or SDF-1{alpha} Ab was mixed with biotinylated rhRANTES or rhSDF-1 and incubated for 15 minutes at room temperature. As negative binding control, an identical sample of cells was stained with a biotinylated soybean trypsin inhibitor, a membrane-binding molecule irrelevant for animal cells. The KG1a cell line, obtained from a patient with acute myeloblastic leukemia, was used as positive control.

Western Blotting
Unstimulated and TNF{alpha}-stimulated BM MCS confluent layers were detached in PBS-EDTA (1 mM) and then lysed with lysing buffer (Sigma-Aldrich). Protein concentration was measured using the Bradford protein assay reagent. Proteins were denatured by boiling for 3 minutes in the presence of ß-mercaptoethanol. Proteins were then separated in 12% (wt/vol) SDS-polyacrylamide gel. Fifteen micrograms of proteins was loaded per lane. After separation, proteins were electrophoretically transferred onto polyvinylidene difluoride membrane. Nonspecific binding was blocked by incubation with 5% (wt/vol) dried milk in TTBS buffer (10 mM Tris/HCl [pH 8.3], 0.05% Tween-20, and 150 mM NaCl) for 60 minutes at room temperature. Membranes were then incubated with the following primary antibodies: rabbit anti-human polyclonal Abs directed against CCR1 and CCR2 and mouse anti-human McAb directed against CCR4. All Abs were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, http://www.scbt.com) or from R&D Systems. Secondary antibodies were mouse or rabbit anti-mouse IgG conjugated to horseradish peroxidase (Bio-Rad, Hercules, CA, http://www.bio-rad.com). Revelation by chemiluminescence was performed with the ECLplus Western blotting detection kit (Amersham Biosciences, Saclay-Orsay, France, http://www.amersham.com), and acquisition was performed with Chemi-Smart 2000 using Chemocapt software (Vilber Lourmat, Marne-la-Vallée, France, http://www.vilber.com). A lysate of total white blood cells was used as positive control.

TaqMan Real-Time Reverse Transcriptase-Polymerase Chain Reaction
P2 confluent layers of unstimulated and stimulated BM MSCs were lysed directly in the culture dishes, and total RNA was isolated using the RNeasy kit (Qiagen, Chatsworth, CA, http://www1.qiagen.com). TaqMan low density arrays (TLDAs) (Micro Fluidic Cards; Applied BioSystems, Foster City, CA, http://www.appliedbiosystems.com) were used in a two-step reverse transcription (RT)-PCR process. First-strand cDNA was synthesized from 3 µg of total RNA using the High-Capacity cDNA Archive Kit (Applied Biosystems). PCRs were then carried out in Micro Fluidic Cards using the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems). We studied 44 genes related to cell migration, including 22 chemokine, 12 cytokine, and 10 protease and inhibitor genes. The probes were labeled with the fluorescent reporter dye 6-carboxyfluorescein (FAM; Applera Corp., Norwalk, CT, http://www.applera.com) on the 5'-end and with nonfluorescent quencher on the 3'-end. cDNA (100 ng) combined with 1x TaqMan Universal Master Mix (Applied Biosystems) was loaded into each well. Micro Fluidic Cards were thermal cycled at 50°C for 2 minutes and 94.5°C for 10 minutes, followed by 40 cycles at 97°C for 30 seconds and 59.7°C for 1 minute. Data were collected with instrument spectral compensations by the Applied Biosystems SDS 2.2.1 software and analyzed using the threshold cycle (CT) relative quantification method. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) reference gene was used for normalizing the data. The 2–{Delta}·CT that corresponds to the ratio of each gene expression to GAPDH expression was calculated. The data presented are mean ± SEM from three independent experiments.

Statistics
Statistical differences between two groups were evaluated by the nonparametric Mann-Whitney U test for independent values and by the Wilcoxon t test for paired values (using StatView software; SAS Institute Inc., Cary, NC, http://www.statview.com). Differences were considered significant when the p value was <.05.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures of Potential...
 Acknowledgments
 References
 
Before performing specific studies, we verified that culture-amplified cells were negative for CD34, CD45, and CD14 surface markers and positive for CD73, CD90, and CD106 and gave rise to adipocytes, osteoblasts, and chondrocytes when placed in adequate differentiating conditions to show their multipotentiality (data not shown).

Growth Factors Are More Potent Chemotactic Agents Than Chemokines for Unstimulated BM MSCs
To investigate the capacity of GFs and chemokines to induce migration of BM MSCs, we tested seven GFs and nine chemokines (CC, CXC, and CX3C chemokines) using an in vitro migration model. As shown in Figure 1A, GFs were better chemoattractants than chemokines. The highest and significant (p < .01) chemotactic activity was observed with PDGF-AB and IGF-1; a lesser but significant (p < .01) activity was observed with EGF and HGF. Values obtained with PDGF-AB were even superior to values with medium supplemented with 30% FCS (positive control). Among chemokines, only RANTES, MDC, and SDF-1 had significant (p < .05) chemotactic activity. However, chemokine values did not reach those obtained with PDGF-AB or IGF-1.


Figure 1
View larger version (26K):
[in this window]
[in a new window]

  		Figure 1. Migration capacity of Unst and TNF{alpha}-primed bone marrow (BM) MSCs toward growth factors (GFs) and chemokines. In vitro migration of BM MSC took place through gelatin-coated transwells. After overnight incubation, migrated cells that remained on the lower face of the filters were stained and counted as indicated in Materials and Methods. The following concentrations of soluble factors were used: 10 ng/ml VEGF, 10 ng/ml EGF, 50 ng/ml HGF, 30 ng/ml IGF-1, 10 ng/ml PDGF-AB, 10 ng/ml FGF-2, 10 ng/ml ANGPT-1, 100 ng/ml MCP-1, 50 ng/ml MIP-1{alpha}, 150 ng/ml RANTES, 100 ng/ml Eotaxin-1, 100 ng/ml Eotaxin-2, 100 ng/ml MDC, 300 ng/ml Fractalkine, 50 ng/ml GRO{alpha}, 150 ng/ml SDF-1. (A): Migration of Unst BM MSCs. Different recombinant factors were compared with migration medium alone (neg control). The same medium supplemented with 30% FCS served as positive control. Columns represent mean percentage of migrated cells related to negative control, and bars represent SEM from seven independent experiments. *, p < .05; **, p < .01. (B): Migration of Unst and prestimulated BM MSCs. BM MSCs were prestimulated for 24 hours with 1 ng/ml TNF{alpha} or IL1ß and allowed to migrate toward medium alone supplemented or not with 30% FCS. Columns represent mean total Nb (±SEM) of migrated cells per filter from three independent experiments. #, Migration values of prestimulated MSCs were higher than values of Unst MSCs in the three experiments. (C): Migration of TNF{alpha}-stimulated BM MSCs toward purified chemotactic factors. BM MSCs were prestimulated for 24 hours with 1 ng/ml TNF{alpha} or not and were allowed to migrate toward medium alone or medium supplemented with recombinant factors. Columns represent mean total Nb (±SEM) of migrated cells per filter from three independent experiments. #, Migration values of TNF{alpha}-stimulated MSCs were higher than values of Unst MSCs in the three experiments. (D): May-Grünwald-Giemsa-stained membrane. Representative stained filters of Unst MSCs toward medium alone and of Unst and TNF{alpha}-stimulated MSCs toward RANTES. Abbreviations: ANGPT-1, angiopoietin-1; EGF, epidermal growth factor; FCS, fetal calf serum; FGF, fibroblast growth factor; HGF, hepatocyte growth factor; IGF, insulin-like growth factor; IL, interleukin; MDC, macrophage-derived chemokine; Nb, number; Neg Ctrl, negative control; PDGF, platelet-derived growth factor; SDF, stromal-derived factor; TNF{alpha}, tumor necrosis factor {alpha}; Unst, unstimulated; VEGF, vascular endothelial growth factor.

 
TNF{alpha}-Primed BM MSCs Show Enhanced Migration Capacity
The "spontaneous" migration capacity of BM MSCs in the presence of medium alone (without FCS, GFs, or chemokines) was low (226 ± 100 cells per insert); it increased in presence of 30% FCS (1336 ± 121 cells per insert) (Fig. 1B). We hypothesized that inflammatory mediators could influence MSC migration. We therefore pretreated BM MSCs with TNF{alpha} (1 ng/ml) or IL1ß (1 ng/ml) for 24 hours before the migration assay. Only TNF{alpha} was found to clearly modify MSC migration by increasing both spontaneous (by 71%) and FCS-induced (by 170%) MSC migration. These results prompted us to evaluate the effect of TNF{alpha}-stimulation on MSC migration induced by purified cytokines including GFs and chemokines.

TNF{alpha}-primed BM MSCs showed increased migration capacity toward the chemokines RANTES, SDF-1, and MDC (Fig. 1C) by 415%, 267%, and 181%, respectively. Interestingly, the absolute number of migrated MSCs with RANTES after TNF{alpha}-stimulation was comparable to that of unstimulated MSCs with IGF-1. On the contrary, in the presence of GFs, the migration capacity of TNF{alpha}-primed BM MSCs was not significantly different from that of unstimulated cells, except for Ang-1.

BM MSCs Express a Number of Growth Factor and Chemokine Receptors that Are Modulated by TNF{alpha} Stimulation
According to the chemotactic response of BM MSCs to GFs and chemokines, we examined by flow cytometry (Fig. 2A) and Western blotting (Fig. 2B) the membrane expression of relevant receptors. We found high-level expression of PDGF-R{alpha}, PDGF-Rß, FGF-R2, EGF-R, TIE-2, and Ang-1 receptor and low/intermediate levels of HGF-R (c-Met) and IGF1-R. TNF-R1 and TNF-R2 were found expressed at a low level (data not shown). Unstimulated BM MSCs expressed the following chemokine receptors: among CC receptors, CCR3 at a high level and CCR2, CCR4, and CCR5 at a low/intermediate level; among CXC receptors, only CXCR4 and CXCR5 at a high level, whereas CXCR1 and CXCR2 were not detected. TNF{alpha}-stimulation of BM MSCs enhanced membrane expression only for FGF-R2 and CCR3, leading to an increase in signal/noise ratio by 214% and 231%, respectively.


Figure 2
View larger version (31K):
[in this window]
[in a new window]

  		Figure 2. Protein expression of growth factor and chemokine receptors on Unst and tumor necrosis factor {alpha} (TNF{alpha})-primed BM MSC. (A): Flow cytometry. Passage 2 (P2) BM MSCs were detached using trypsin and incubated with specific or isotypic negative control antibody (Ab). Live 7-aminoactinomycin D– cells were analyzed for fluorescence. The results presented are from one representative experiment of three. Black and red curves represent the distribution of fluorescence intensity with specific Ab on Unst and TNF{alpha}-Stm MSCs, respectively, and gray areas represent that with negative control Ab. Values indicated in each panel are the ratio of mean fluorescence intensity of Unst BM MSCs with specific Abs and negative control Ab. (B): Western blotting. Proteins from Unst and TNF{alpha}-Stm P2 BM MSCs were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane. The blot was incubated with Abs against CCR1, CCR2, or CCR4. WBC lysates were used as positive control. (C): RANTES and SDF-1{alpha} binding assays. Cells were preincubated with biotinylated RANTES and SDF-1 or with irrelevant vegetal cell molecule as negative binding control. Membrane binding of cytokines was revealed by incubation with avidin-fluorescein isothiocyanate (as described in Materials and Methods). KG1a cells were used as positive control cells. a, Histogram plots show the level of RANTES and SDF-1 bound to KG1a cells (black curve) as compared with negative control cytokine (gray area). The specificity of the binding assays was measured after preincubation with blocking Abs against RANTES and SDF-1 (blue curve). b, Histogram plots show the level of RANTES and SDF-1 bound to Unst (black curve) and TNF{alpha}-Stm MSCs (red curve) as compared with negative control cytokine (gray area). Abbreviations: BM, bone marrow; EGF-R, epidermal growth factor receptor; FGF-R, fibroblast growth factor receptor; HGF-R, hepatocyte growth factor receptor; IGF1-R, insulin-like growth factor 1 receptor; PDGF-R, platelet-derived growth factor receptor; S/N, mean signal/noise ratios from the three experiments; Stm, stimulated; Unst, unstimulated; WBC, white blood cell.

 
Because of the low level or the lack of expression of CCR1, CCR2, and CCR4 found by flow cytometry, we also evaluated these receptors by Western blotting. CCR2 and CCR4 were clearly detected, and the band intensity was increased in TNF{alpha}-primed cells.

Considering the redundancy of chemokines for their receptors, we decided to complete these studies on chemokine receptors by functional assays that evaluated the binding of labeled RANTES and SDF-1 on MSCs. As shown in Figure 2C, both RANTES and SDF-1 bound to unstimulated BM MSCs. After TNF{alpha} stimulation, an increase in binding was observed for RANTES but not for SDF-1. These findings are consistent with the clear TNF{alpha}-enhanced MSC migration toward RANTES and the increased expression of CCR3 (one of the RANTES receptors) observed in TNF{alpha}-primed MSCs.

mRNAs for Growth Factors, Chemokines, and Proteases Are Differentially Expressed by Unstimulated and TNF{alpha}-Stimulated BM MSCs
Although TNF{alpha}-enhanced BM MSC migration could be explained by the increase in cytokine receptor expression, we investigated whether the modulation of soluble factors could also contribute to this phenomenon. We studied the expression pattern of a total of 44 genes known to be implicated in cell migration: 22 CC, CXC, CX3C, and XC chemokines; 12 cytokines (11 GFs and TNF{alpha}); and 10 proteases and inhibitors. We used TLDAs, which allow the level of mRNA to be quantified compared with a housekeeping gene (GAPDH). Several genes encoding chemokines (Fig. 3A) were found to be expressed constitutively at a high level (MCP-1, GRO{alpha}, IL8, and SDF-1) or at a low/intermediate level (RANTES, MCP-3, Eotaxin-3, Fractalkine, GROß, ENA-78, IP-10, and I-TAC). TNF{alpha} stimulation (a) induced the expression of I-309, MIP-1{alpha}, Eotaxin-1, TARC, and MIG; (b) increased the expression of MCP-1, RANTES, Fractalkine, GRO{alpha}, GROß, ENA-78, IL8, IP-10, and i-TAC; and (c) did not modulate the expression of MCP-3, Eotaxin-3, and SDF-1. Some chemokine genes were not expressed at all (MDC, Eotaxin-2, TECK, BCA-1, and Lymphotactin).


Figure 3
View larger version (16K):
[in this window]
[in a new window]

  		Figure 3. mRNA expression of chemokines and growth factors on unstimulated and TNFa-stimulated bone marrow MSCs. Columns represent mean values (and bars represent the SEM) of three separated experiments assessed by TaqMan low density arrays, in black for unstimulated MSCs and in white for TNFa-stimulated MSCs. Twenty-two chemokines, 12 growth factors, and 10 proteases and inhibitors were evaluated. Expression level of each gene is represented by 2–{Delta}CT, where {Delta}CT = CT (gene X) – CT (GAPDH). #, Values of TNFa-stimulated MSCs were higher than values of unstimulated MSCs in the three experiments. Abbreviations: EGF, epidermal growth factor; FGF, fibroblast growth factor; HGF, hepatocyte growth factor; IGF, insulin-like growth factor; IL, interleukin; MMP, matrix metalloproteinase; PDGFa, platelet-derived growth factor {alpha}; PDGFb, platelet-derived growth factor ß; TNFa, tumor necrosis factor {alpha}; VEGF, vascular endothelial growth factor.

 
We detected mRNAs at intermediate/high levels for all GF genes assayed (Fig. 3B), except those for PDGF-B and TNF{alpha}, which were expressed only after TNF{alpha} stimulation.

Proteases are well known to play a critical role in cell migration, because of their ability to degrade extracellular matrix components or locally produced chemokines. We therefore investigated the mRNA expression of some proteases, such as matrix metalloproteinases (MMPs), in BM MSC migration, particularly after TNF{alpha} stimulation (Fig. 3C). MMP-2, its activator MMP-14 (MT1-MMP), and the inhibitors TIMP-1 and TIMP-2 were found constitutively expressed at a high level in BM MSCs. A similar expression pattern was seen for CD26/dipeptidyl peptidase IV (DPPIV), an ectoenzyme able to inactivate a number of chemokines (including SDF-1) by limited cleavage of the molecule [32]. In contrast, the mRNA expression found at intermediate/low levels for MMP-1, -3, -7, and -13 was increased to a high level after TNF{alpha} stimulation. MMP-9 was the only MMP not expressed at steady state or induced by TNF{alpha} stimulation.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures of Potential...
 Acknowledgments
 References
 
This study shows that BM MSCs are able to migrate in vitro in response to a large set of chemotactic factors, including both GFs and chemokines. The GFs PDGF-AB and IGF-1 appear to be the most potent factors, whereas some chemokines (RANTES, MDC, and SDF-1) have limited effect. However, preincubation with the inflammatory cytokine TNF{alpha} leads to increased MSC migration toward chemokines, especially RANTES. The migration capacity of MSCs clearly stands in contrast to that of HSCs whose migration is induced mainly by a single chemokine, SDF-1 [33], whereas hematopoietic GFs display little or no chemotactic activity [34].

We tested seven GFs known to be produced by mesenchymal cells and known for their promigratory, proliferating, or differentiating activity on mesenchymal cells. In our study, the two most chemoattractive GFs were PDGF-AB and IGF-1, which confirms reports by Fiedler et al. [28, 35]. The three other GFs with chemoattractive capacity were HGF, EGF, and Ang-1. HGF and EGF are already reported as chemoattractants for MSCs [29, 30, 36]. Ang-1 is a crucial chemotactic factor for endothelial cells through its receptor Tie-2 [37]. Recently, HSCs expressing Tie-2 have been shown to respond to Ang-1 by inducing cobblestone area formation and maintaining long-term repopulating activity [38]. In the present study, we show, for the first time to the best of our knowledge, that human (Hu) BM-derived MSCs express Tie-1 and are chemoattracted by Ang-1 under proinflammatory conditions (i.e., in the presence of TNF{alpha}). This result is in contrast with the negative results we observed for VEGF, another potent angiogenic factor, underlining a major difference between MSCs and endothelial cells. As expected from the promigratory response, MSCs expressed, at a moderate to high level, receptors for PDGF-AB, IGF-1, HGF, and EGF. MSC migration capacity to these major chemotactic GFs was unchanged after TNF{alpha} exposure, as was the membrane receptor expression.

The chemotactic activity of chemokines on Hu BM MSCs appeared clearly less efficient than that of GFs, with only three of the nine chemokines tested displaying a significant activity (RANTES/CCL5, SDF-1/CXCL12, and MDC/CCL22). The most active was MDC, with induced migration levels equivalent to those obtained with the less active GFs (EGF and HGF). RANTES and SDF-1 showed a lesser, although significant, effect. Consistent with these results, we detected on MSCs the receptors for RANTES (CCR3, CCR4, and CCR5, but not CCR1), MDC (CCR4), and SDF-1 (CXCR4). Although found at low levels by flow cytometry, CCR2 and CCR4 expression was more evident using Western blotting, probably because of the concomitant detection of the intracellular pool. In contrast, CCR1 was not detected by either technique. The most striking finding was the dramatic increase, after TNF{alpha} stimulation, in MSC sensitivity to RANTES, MDC, and SDF-1. In TNF{alpha}-primed cells, we observed not only increased migration in response to RANTES and MDC but also increased expression of the receptors CCR3 and CCR4, using flow cytometry or Western blotting. The functional relevance of these observations was confirmed by experiments showing that the membrane binding of RANTES was increased in TNF{alpha}-stimulated cells. In contrast, TNF{alpha}-stimulated migration of MSCs to SDF-1 was not associated with either increased membrane expression of its receptor CXCR4 or increased SDF-1 surface binding, a discrepancy already reported for dendritic cells submitted to a similar regimen [39]. TNF{alpha} might modify the MSC sensitivity to SDF-1 by modulating CXCR4 signal transduction without affecting receptor expression, as described after inhibition of the protein kinase C pathway [40]. Some reports have recently pointed out the potential role of the SDF-1/CXCR4 axis on MSC migration [25–27, 41]. Our study not only confirms these reports but also indicates that the promigratory activity of this chemokine is increased in TNF{alpha}-primed cells, suggesting an increased role of SDF-1 under inflammatory conditions in which TNF{alpha} can be overexpressed. The activity of RANTES on BM MSCs has been reported [25]; our results clearly indicate that it is, together with SDF-1, the most efficient chemokine after TNF{alpha}-stimulation. Finally, we show, for the first time to the best of our knowledge, that MDC, a chemokine active on mature hematopoietic cells [42], is also a potent chemoattractant for Hu BM MSCs, even displaying the strongest activity among the chemokines tested on steady-state cells.

Using large-scale quantitative RT-PCR, we evaluated, in TNF{alpha}-primed cells and in unstimulated cells, the expression at the mRNA level of a large panel of factors, including not only chemokines (from the CC, CXC, XC, and CX3C families) and cytokines but also proteases and inhibitors. Of the 22 chemokines tested, only 5 were not expressed (including MDC), 14 were induced or increased after TNF{alpha} treatment (including MCP-1 and RANTES), and no change was observed for 3 (including SDF-1). Of the 11 GFs tested, 10 were constitutively expressed (with TNF{alpha}-increased levels only for IGF-1), and only 1 was induced (PDGF-B). Considering these results, we may have underevaluated the observed promigratory activity of certain chemokines and GFs because of the autocrine MSC production leading to the reduction of the imposed concentration gradient. Proteases, such as MMPs and CD26/DPPIV, are able to favor cell locomotion and tissue reconstitution by breaking down the extracellular matrix but also by degrading locally a number of chemokines [43]. In particular, MMP-2 and MT1-MMP (MMP-14) have been reported to be involved in MSC migration induced by PDGF [44], HGF, or SDF-1 [41], whereas MT1-MMP cellular expression has been shown very recently to be upregulated by inflammatory cytokines, including TNF{alpha} [45]. In the present study, we showed that MMP-1, -2, -3, -13, MT1-MMP, CD26, and the inhibitors TIMP-1 and -2 are constitutively expressed by BM MSCs, in contrast to MMP-7 and -9. TNF{alpha} treatment strongly enhances the expression of MMP-1, -3, and -13 and induces that of MMP-9, as found by other authors [45]. Thus, MSC migration might be favored by increased local concentrations of collagenases and gelatinases, particularly in inflammatory situation.

Our results on chemotactic factors, either GFs or chemokines, are summarized in Table 1. The chemotactic factors identified here are particularly relevant for MSC homing and tissue repair, since tissue damage has been associated with local increase in mediators, such as those reported for SDF-1 or HGF [26, 46, 47]. In vivo use of chemotactic factors for MSCs may be of clinical interest, and they now need to be tested in animal models. On the one hand, local administration might improve MSC homing to specific tissues, as recently reported with IGF-1, HGF, or PDGF after myocardial damage [48], and, on the other hand, systemic administration might trigger endogenous MSC mobilization for tissue repair, avoiding ex vivo amplification. In addition, the local or systemic production of inflammatory mediators might influence not only MSC migration, as shown in this study and another one [45], but also MSC proliferation and differentiation [49] and MSC engraftment [50].


View this table:
[in this window]
[in a new window]

  		Table 1. Summary of the sensitivity pattern of human bone marrow MSCs to chemotactic factors compared to relevant membrane receptor expression

 
In conclusion, our data indicate that the migration capacity of BM MSCs is under the control of a large range of receptor tyrosine kinase receptor GFs and CC and CXC chemokines. These chemokines appear mostly effective on TNF{alpha}-primed cells, suggesting that the mobilization of MSCs and their subsequent homing to injured tissues may depend on the systemic and local inflammatory state. This parameter has to be taken into account in cell therapy protocols using BM MSCs.


   DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures of Potential...
 Acknowledgments
References
 
We are grateful to Elfi Ducrocq and Marie-Christine Bernard for expert technical assistance. This work was supported by grants from the European Integrated projects FIRST (contract no. 503436) and GENOSTEM (contract no. 503161), from the French Cancéropôle Grand-Ouest network, and from the Committees of Cher and Vendée of the French League against Cancer.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures of Potential...
 Acknowledgments
 References
 

  1. Dennis JE, Charbord P. Origin and differentiation of human and murine stroma. STEM CELLS 2002;20:205–214.[Abstract/Free Full Text]

  2. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997;276:71–74.[Abstract/Free Full Text]

  3. Caplan AI. Mesenchymal stem cells. J Orthop Res 1991;9:641–650.[CrossRef][Medline]

  4. Liechty KW, MacKenzie TC, Shaaban AF et al. Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep. Nat Med 2000;6:1282–1286.[CrossRef][Medline]

  5. Dezawa M, Ishikawa H, Itokazu Y et al. Bone marrow stromal cells generate muscle cells and repair muscle degeneration. Science 2005;309:314–317.[Abstract/Free Full Text]

  6. Kawada H, Fujita J, Kinjo K et al. Nonhematopoietic mesenchymal stem cells can be mobilized and differentiate into cardiomyocytes after myocardial infarction. Blood 2004;104:3581–3587.[Abstract/Free Full Text]

  7. Kopen GC, Prockop DJ, Phinney DG. Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc Natl Acad Sci U S A 1999;96:10711–10716.[Abstract/Free Full Text]

  8. Sanchez-Ramos J, Song S, Cardozo-Pelaez F et al. Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol 2000;164:247–256.[CrossRef][Medline]

  9. Chagraoui J, Lepage-Noll A, Anjo A et al. Fetal liver stroma consists of cells in epithelial-to-mesenchymal transition. Blood 2003;101:2973–2982.[Abstract/Free Full Text]

  10. Lee KD, Kuo TK, Whang-Peng J et al. In vitro hepatic differentiation of human mesenchymal stem cells. Hepatology 2004;40:1275–1284.[CrossRef][Medline]

  11. Thomas ED. The use and potential of bone marrow allograft and whole-body irradiation in the treatment of leukemia. Cancer 1982;50:1449–1454.[CrossRef][Medline]

  12. Li Y, Chen J, Wang L et al. Intracerebral transplantation of bone marrow stromal cells in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease. Neurosci Lett 2001;316:67–70.[CrossRef][Medline]

  13. Pereira RF, O'Hara MD, Laptev AV et al. Marrow stromal cells as a source of progenitor cells for nonhematopoietic tissues in transgenic mice with a phenotype of osteogenesis imperfecta. Proc Natl Acad Sci U S A 1998;95:1142–1147.[Abstract/Free Full Text]

  14. NiedYwiedzki T, Dabrowski Z, Miszta H et al. Bone healing after bone marrow stromal cell transplantation to the bone defect. Biomaterials 1993;14:115–121.[CrossRef][Medline]

  15. Almeida-Porada G, Porada CD, Tran N et al. Cotransplantation of human stromal cell progenitors into preimmune fetal sheep results in early appearance of human donor cells in circulation and boosts cell levels in bone marrow at later time points after transplantation. Blood 2000;95:3620–3627.[Abstract/Free Full Text]

  16. Koç ON, Gerson SL, Cooper BW et al. Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol 2000;18:307–316.[Abstract/Free Full Text]

  17. Le Blanc K, Rasmusson I, Sundberg B et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet 2004;363:1439–1441.[CrossRef][Medline]

  18. Rochefort GY, Vaudin P, Bonnet N et al. Influence of hypoxia on the domiciliation of mesenchymal stem cells after infusion into rats: Possibilities of targeting pulmonary artery remodeling via cells therapies? Respir Res 2005;6:125.[Medline]

  19. Chapel A, Bertho JM, Bensidhoum M et al. Mesenchymal stem cells home to injured tissues when co-infused with hematopoietic cells to treat a radiation-induced multi-organ failure syndrome. J Gene Med 2003;5:1028–1038.[CrossRef][Medline]

  20. Devine SM, Bartholomew AM, Mahmud N et al. Mesenchymal stem cells are capable of homing to the bone marrow of non-human primates following systemic infusion. Exp Hematol 2001;29:244–255.[CrossRef][Medline]

  21. Devine SM, Cobbs C, Jennings M et al. Mesenchymal stem cells distribute to a wide range of tissues following systemic infusion into nonhuman primates. Blood 2003;101:2999–3001.[Abstract/Free Full Text]

  22. Fouillard L, Bensidhoum M, Bories D et al. Engraftment of allogeneic mesenchymal stem cells in the bone marrow of a patient with severe idiopathic aplastic anemia improves stroma. Leukemia 2003;17:474–476.[CrossRef][Medline]

  23. Rochefort GY, Delorme B, Lopez A et al. Multipotential mesenchymal stem cells are mobilized into peripheral blood by hypoxia. STEM CELLS 2006;24:2202–2208.[Abstract/Free Full Text]

  24. Peled A, Petit I, Kollet O et al. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4. Science 1999;283:845–848.[Abstract/Free Full Text]

  25. Honczarenko M, Le Y, Swierkowski M et al. Human bone marrow stromal cells express a distinct set of biologically functional chemokine receptors. STEM CELLS 2006;24:1030–1041.[Abstract/Free Full Text]

  26. Ji JF, He BP, Dheen ST et al. Interactions of chemokines and chemokine receptors mediate the migration of mesenchymal stem cells to the impaired site in the brain after hypoglossal nerve injury. STEM CELLS 2004;22:415–427.[Abstract/Free Full Text]

  27. Sordi V, Malosio ML, Marchesi F et al. Bone marrow mesenchymal stem cells express a restricted set of functionally active chemokine receptors capable of promoting migration to pancreatic islets. Blood 2005;106:419–427.[Abstract/Free Full Text]

  28. Fiedler J, Roderer G, Gunther KP et al. BMP-2, BMP-4, and PDGF-BB stimulate chemotactic migration of primary human mesenchymal progenitor cells. J Cell Biochem 2002;87:305–312.[CrossRef][Medline]

  29. Forte G, Minieri M, Cossa P et al. Hepatocyte growth factor effects on mesenchymal stem cells: Proliferation, migration, and differentiation. STEM CELLS 2006;24:23–33.[Abstract/Free Full Text]

  30. Neuss S, Becher E, Woltje M et al. Functional expression of HGF and HGF receptor/c-met in adult human mesenchymal stem cells suggests a role in cell mobilization, tissue repair, and wound healing. STEM CELLS 2004;22:405–414.[Abstract/Free Full Text]

  31. Delorme B, Charbord P. Culture and characterisation of human bone marrow mesenchymal stem cells. In: Hauser H, Fussenegger M, eds. Methods in Molecular Medicine: Tissue Engineering. 2nd ed. Totowa, NJ: Humana Press, Inc., in press.

  32. Lambeir AM, Proost P, Durinx C et al. Kinetic investigation of chemokine truncation by CD26/dipeptidyl peptidase IV reveals a striking selectivity within the chemokine family. J Biol Chem 2001;276:29839–29845.[Abstract/Free Full Text]

  33. Wright DE, Wagers AJ, Gulati AP et al. Physiological migration of hematopoietic stem and progenitor cells. Science 2001;294:1933–1936.[Abstract/Free Full Text]

  34. Okumura N, Tsuji K, Ebihara Y et al. Chemotactic and chemokinetic activities of stem cell factor on murine hematopoietic progenitor cells. Blood 1996;87:4100–4108.[Abstract/Free Full Text]

  35. Fiedler J, Brill C, Blum WF et al. IGF-I and IGF-II stimulate directed cell migration of bone-marrow-derived human mesenchymal progenitor cells. Biochem Biophys Res Commun 2006;345:1177–1183.[CrossRef][Medline]

  36. Tamama K, Fan VH, Griffith LG et al. Epidermal growth factor as a candidate for ex vivo expansion of bone marrow-derived mesenchymal stem cells. STEM CELLS 2006;24:686–695.[Abstract/Free Full Text]

  37. Witzenbichler B, Maisonpierre PC, Jones P et al. Chemotactic properties of angiopoietin-1 and -2, ligands for the endothelial-specific receptor tyrosine kinase tie2. J Biol Chem 1998;273:18514–18521.[Abstract/Free Full Text]

  38. Arai F, Hirao A, Ohmura M et al. Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell 2004;118:149–161.[CrossRef][Medline]

  39. Vecchi A, Massimiliano L, Ramponi S et al. Differential responsiveness to constitutive vs. inducible chemokines of immature and mature mouse dendritic cells. J Leukoc Biol 1999;66:489–494.[Abstract]

  40. Petit I, Goichberg P, Spiegel A et al. Atypical PKC-zeta regulates SDF-1-mediated migration and development of human CD34+ progenitor cells. J Clin Invest 2005;115:168–176.[CrossRef][Medline]

  41. Son BR, Marquez-Curtis LA, Kucia M et al. Migration of bone marrow and cord blood mesenchymal stem cells in vitro is regulated by stromal-derived factor-1-CXCR4 and hepatocyte growth factor-c-met axes and involves matrix metalloproteinases. STEM CELLS 2006;24:1254–1264.[Abstract/Free Full Text]

  42. Godiska R, Chantry D, Raport CJ et al. Human macrophage-derived chemokine (MDC), a novel chemoattractant for monocytes, monocyte-derived dendritic cells, and natural killer cells. J Exp Med 1997;185:1595–1604.[Abstract/Free Full Text]

  43. Zabel BA, Zuniga L, Ohyama T et al. Chemoattractants, extracellular proteases, and the integrated host defense response. Exp Hematol 2006;34:1021–1032.[CrossRef][Medline]

  44. Robbins JR, McGuire PG, Wehrle-Haller B et al. Diminished matrix metalloproteinase 2 (MMP-2) in ectomesenchyme-derived tissues of the patch mutant mouse: Regulation of MMP-2 by PDGF and effects on mesenchymal cell migration. Dev Biol 1999;212:255–263.[CrossRef][Medline]

  45. Ries C, Egea V, Karow M et al. MMP-2, MT1-MMP, and TIMP-2 are essential for the invasive capacity of human mesenchymal stem cells: Differential regulation by inflammatory cytokines. Blood 2007; [Epub ahead of print].

  46. Kollet O, Shivtiel S, Chen YQ et al. HGF, SDF-1, and MMP-9 are involved in stress-induced human CD34+ stem cell recruitment to the liver. J Clin Invest 2003;112:160–169.[CrossRef][Medline]

  47. Askari AT, Unzek S, Popovic ZB et al. Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy. Lancet 2003;362:697–703.[CrossRef][Medline]

  48. Urbanek K, Rota M, Cascapera S et al. Cardiac stem cells possess growth factor-receptor systems that after activation regenerate the infarcted myocardium, improving ventricular function and long-term survival. Circ Res 2005;97:663–673.[Abstract/Free Full Text]

  49. Girgenrath M, Weng S, Kostek CA et al. TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration. EMBO J 2006;25:5826–5839 13.[CrossRef][Medline]

  50. Coyne MA, Woodbury D, Black IB. Marrow stromal cells transplanted to the adult brain are rejected by an inflammatory response and transfer donor labels to host neurons and glia. STEM CELLS 2006;24:2483–2492.[Abstract/Free Full Text]




This article has been cited by other articles:


Home page
 Stem CellsHome page
A. M. Sonabend, I. V. Ulasov, M. A. Tyler, A. A. Rivera, J. M. Mathis, and M. S. Lesniak
Mesenchymal Stem Cells Effectively Deliver an Oncolytic Adenovirus to Intracranial Glioma
Stem Cells, March 1, 2008; 26(3): 831 - 841.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page
S. L. Tomchuck, K. J. Zwezdaryk, S. B. Coffelt, R. S. Waterman, E. S. Danka, and A. B. Scandurro
Toll-Like Receptors on Human Mesenchymal Stem Cells Drive Their Migration and Immunomodulating Responses
Stem Cells, January 1, 2008; 26(1): 99 - 107.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
A. H. Klopp, E. L. Spaeth, J. L. Dembinski, W. A. Woodward, A. Munshi, R. E. Meyn, J. D. Cox, M. Andreeff, and F. C. Marini
Tumor Irradiation Increases the Recruitment of Circulating Mesenchymal Stem Cells into the Tumor Microenvironment
Cancer Res., December 15, 2007; 67(24): 11687 - 11695.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page
G. Chamberlain, J. Fox, B. Ashton, and J. Middleton
Concise Review: Mesenchymal Stem Cells: Their Phenotype, Differentiation Capacity, Immunological Features, and Potential for Homing
Stem Cells, November 1, 2007; 25(11): 2739 - 2749.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2007-0054v1
25/7/1737    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ponte, A. L.
Right arrow Articles by Domenech, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ponte, A. L.
Right arrow Articles by Domenech, J.

HOME HELP FEEDBACK