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


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
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 Ji, J. F.
Right arrow Articles by Tay, S. S. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ji, J. F.
Right arrow Articles by Tay, S. S. W.

Stem Cells 2004;22:415-427 www.StemCells.com
© 2004 AlphaMed Press

Interactions of Chemokines and Chemokine Receptors Mediate the Migration of Mesenchymal Stem Cells to the Impaired Site in the Brain After Hypoglossal Nerve Injury

Jun Feng Ji, Bei Ping He, S. Thameem Dheen, Samuel Sam Wah Tay

Department of Anatomy, Faculty of Medicine, National University of Singapore, Singapore

Key Words. Mesenchymal stem cells • Hypoglossal nerve • Chemokines • Chemokine receptors • Transplantation • Lateral ventricle

Samuel Sam Wah Tay, Ph.D., Department of Anatomy, Faculty of Medicine, National University of Singapore, Lower Kent Ridge Road, Singapore 117597. Telephone: 65-68743210; Fax: 65-67787643; e-mail: anttaysw{at}nus.edu.sg


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mesenchymal stem cells (MSCs), cultured ex vivo, recently were shown to be able to migrate into sites of brain injuries when transplanted systemically or locally, suggesting that MSCs possess migratory capacity. However, the mechanisms underlying the migration of these cells remain unclear. In this study, we examined the role of some chemokines and their receptors in the trafficking of rat MSCs (rMSCs) in a rat model of left hypoglossal nerve injury. rMSCs transplanted into the lateral ventricles of the rat brain migrated to the avulsed hypoglossal nucleus, where the expression of chemokines, stromal-cell-derived factor 1 (SDF-1), and fractalkine was observed to be increased. This increase temporally paralleled the migration of rMSCs into the avulsed nucleus at 1 and 2 weeks after operation. It has been found that rMSCs express CXCR4 and CX3CR1, the respective receptors for SDF-1 and fractalkine, and other chemokine receptors, CCR2 and CCR5. Furthermore, in vitro analysis revealed that recombinant human SDF-1 alpha (rhSDF-1{alpha}) and recombinant rat fractalkine (rrfractalkine) induced the migration of rMSCs in a G-protein-dependent manner. Intracerebral injection of rhSDF-1{alpha} has also been shown to stimulate the homing of transplanted rMSCs to the site of injection in the brain. These data suggest that the interactions of fractalkine-CX3CR1 and SDF-1–CXCR4 could partially mediate the trafficking of transplanted rMSCs. This study provides an important insight into the understanding of the mechanisms governing the trafficking of transplanted rMSCs and also significantly expands the potential role of MSCs in cell therapy for brain injuries and diseases.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bone marrow contains precursors for hematopoietic cells and stem-like cells for a variety of nonhematopoietic tissues. The precursors of nonhematopoietic tissues were initially referred to as plastic-adherent cells or colony-forming units fibroblasts because of their ability to adhere to culture dishes and form fibroblast-like colonies [1]. In addition, these cells were referred to as mesenchymal stem cells (MSCs) or mesenchymal progenitor cells because they possess the capacity to differentiate into a variety of nonhematopoietic cells [2]. Moreover, they have been referred to as marrow stromal cells because they arise from the supporting structures in bone marrow and can act as feeder layer for the growth of hematopoietic stem cells in culture [3].

MSCs have been shown to have multipotential capacities to differentiate into osteoblasts, adipocytes, chondrocytes, myoblasts, and myotubes [1, 4–6]. Recent data have pointed to the unexpected ability of both human and rodent MSCs to differentiate into neural cells [7–9]. The evidence has suggested their therapeutic potential to regenerate neural cells in the injured or diseased brain [10].

Interestingly, recent studies from Chopp’s group [11–15] have demonstrated the capability of in vitro propagated rat MSCs (rMSCs) to migrate selectively into damaged areas in the brain after the rMSCs were systemically or locally implanted. In the models of traumatic or ischemic brain injuries, rMSCs administered intravenously, intra-arterially, or intracerebrally preferentially migrated into the region of neurodegeneration in the brain [11–15]. Identification of the molecular signals governing rMSC migration in vivo is of major importance for understanding the MSC-mediated cell therapy for traumas and diseases in the central nervous system (CNS). Although an initial study suggested a role for chemokines in both rMSC and human MSC migration in vitro [16, 17], the signaling pathway relevant to their directed migration still remains unknown. In this study, we attempted to investigate the hypothesis that the interaction between chemokines and their receptors could play important roles in the migration of rMSCs to the impaired sites in the CNS in the model of left hypoglossal nerve avulsion. Unilateral hypoglossal nerve avulsion has been proven to cause severe motoneuronal death in the injured nucleus but no cell death in the contralateral side [18]. Therefore, it would be a simple model to provide a practical comparison between the avulsed and intact nuclei to study the migration of transplanted rMSCs. Our results suggest that the interaction between fractalkine and its receptor CX3CR1 and the interaction of stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4 could partially mediate the trafficking of rMSCs to the impaired nucleus in the brain.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
MSCs Primary Culture
We isolated rMSCs as previously described with some modifications [19]. Briefly, the whole bone marrow plugs were obtained by flushing the bone marrow cavity of the femurs and tibiae of adult Wistar rats with a 21-gauge syringe filled with alpha minimal essential medium (Invitrogen Life Technologies; Carlsbad, CA; http://www.invitrogen.com) supplemented with 15% fetal calf serum (FCS), 2 mM L-glutamine and 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B (Sigma; St. Louis, MO; http://www.sigmaaldrich.com). Cells were passed through a 70 µm cell strainer (BD Bioscience Discovery Labware; Bedford, MA; http://www.bdbiosciences.com) to remove remaining clumps of tissue and then plated in a 75 cm2 flask and incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2. The first medium change was done within 24 hours to remove the nonadherent hematopoietic lineage cells, and the remaining adherent cells were defined as passage zero (P0) cells. For further expanding and processing, the P0 cells at 90% confluency were detached by incubation with 0.25% trypsin (Sigma) and 1 mM EDTA (Sigma) for 5 minutes at 37°C, plated as P1 in 75 cm2 flasks at density of 5,000 cells/cm2, and grown to 90% confluency. All cells used in these studies were P4 or earlier. All procedures involving animals were approved by the Medical Faculty Ethics Committee, National University of Singapore.

In Vitro Differentiation Assay
For osteogenic differentiation [20], we plated detached rMSCs at 1,000 cells/cm2 and grew them to 50%-70% confluency in 5 days. They were then incubated in osteogenic medium containing 10–8 M dexamethasone, 0.2 mM ascorbic acid, and 10 mM ß-glycerolphosphate (Sigma). The medium was replaced every 3–4 days for 21 days. Cultures were washed with phosphate-buffered saline (PBS), fixed in a solution of ice-cold 70% ethanol for 1 hour, and stained for 10 minutes with 1 ml of 40 mM Alizarin red (pH 4.1) (Sigma). For adipogenic differentiation [20], 50%-70% confluent cultures were incubated in complete medium supplemented with 0.5 µM hydrocortisone, 0.5 mM isobutylmethylxanthine, and 60 µM indomethacin (Sigma). The medium was replaced every 3–4 days for 21 days. Cells were washed with PBS, fixed in 10% formalin for 10 min, and stained for 15 minutes with fresh Oil red-O solution (Fisher Scientific; Pittsburgh, PA; http://www.fishersci.com).

Cortical Microglia Primary Culture
For mRNA expression of CXCR4, CCR2, CCR5, and CX3CR1 analysis, we used cultured cortical microglial cells, which have been reported to express CCR2, CCR5, CXCR4, and CX3CR1 mRNA [21–23], as positive control. Microglial cells were isolated from high-density glial cell cultures. Briefly, cells dissociated from the cerebral hemispheres of 3–5-day-old postnatal rats (Wistar strain) were plated at a density of 1.2 x 106 cells/ml in a culture medium consisting of Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% FCS, 0.1 mM nonessential amino acid (Invitrogen), 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B (Sigma). Culture medium was changed after the first 3 days and then twice a week. After 2 weeks, the loosely adherent microglial cells were recovered by shaking. After centrifugation (100 x g for 5 min), viable cells were plated at a final density of 1.0 x 106/well on a six multiwell Falcon plate in complete medium. Nonadherent cells were removed 30 minutes after plating by changing medium. The purity of microglia was assessed immunochemically using OX-42 (1:100; Sera-Lab; Leicestershire, UK; http://www.harlanseralab.co.uk), and anti-glial fibrillary acidic protein (GFAP) (1:800, Santa Cruz Biotechnology; Santa Cruz, CA; http://www.scbt.com). We examined cellular morphology under a phase contrast microscope.

Fluorescent Activated Cell Sorting (FACS) Analysis
Cultured rMSCs were detached with EDTA/trypsin and washed by centrifugation in PBS. The cells were then fixed in 1% methanol at 4°C for 10 minutes and washed with PBS. Nonspecific antigens were blocked by incubating the cells at room temperature for 1 hour in 1% bovine serum albumin (BSA), 0.1% FCS, and 0.1% horse serum. The cell pellet was suspended in 0.5 ml of mouse anti-CD11b (1:100; Serotec; Oxford, UK; http://www.serotec.co.uk) or anti-CD45 (1:100; BD Biosciences Pharmingen; San Diego, CA). After incubation for 40 minutes at 4°C, the cells were washed in PBS and then incubated in 0.5 ml of anti-mouse immunoglobulin G (IgG) labeled with fluorescein isothiocyanate (FITC) (1:200, Sigma) for 1 hour. For chemokine receptor expression analysis, the cells were similarly processed except that the following primary antibodies and secondary antibody were used: goat polyclonal CXCR4 (1:100), CCR2 (1:100), and CCR5 (1:100) (Santa Cruz Biotechnology); rabbit polyclonal CX3CR1 (1:100; Torry Pines Biolabs, Inc.; Houston, TX; http://www.chemokine.com); anti-goat IgG conjugated with Cy3 (1:200; Chemicon International; Temecula, CA; http://www.chemicon.com), and anti-rabbit IgG conjugated with FITC (1:200. Sigma). The cells were then washed in PBS, resuspended in 0.5 ml PBS at concentrations of 2 - 10 x 104 per ml, and assayed in a flow cytometer (FACsort) (Becton-Dickinson; Franklin Lakes, NJ; http://www.bd.com). For an isotype control, nonspecific mouse immunoglobulin (DAKO) was substituted for the primary antibody.

Labeling of rMSCs In Vitro
We washed rMSCs with three volumes of PBS. The cells were incubated in 10 ml of 10 µM 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) (Molecular Probes, Inc.; Eugene, OR; http://www.probes.com) in PBS for 30 minutes at 37°C. The positive labeling was examined using a fluorescent microscope. For transplantation study, labeled rMSCs were detached with trypsin/EDTA and washed and reconstituted in PBS.

Surgical Operation
A total of 20 adult male Wistar rats (weighing about 250 g each) were used in the immunohistochemical study on the expression of chemokines in the hypoglossal nucleus (HN) after left hypoglossal nerve injury. Under deep anesthesia induced by intraperitoneal injection of 7% chloral hydrate (0.6 ml/100 g body weight), the left hypoglossal nerves of the rats were avulsed at the upper-cervical level. Sham-operated and normal animals served as controls. The animals were sacrificed at 1 week or 2 weeks after operation. Each group contained 3 to 5 rats. Before sacrifice, rats were anesthetized with 7% chloral hydrate and subsequently perfused with 4% paraformaldehyde. Serial frozen coronal sections of the brainstem (20 µm thick) were cut using a cryostat for immunohistochemical staining. The chemokine-immunopositive cells in the HN of every fifth section were counted and statistically analyzed using Student’s t-test.

Immunohistochemistry and Immunocytochemistry
Immunostaining was performed on the frozen sections of the brainstem and the cells plated on polylysine-coated coverslips. The sections or cells were blocked with normal rabbit, goat, or horse serum according to the species of the secondary antibodies for 1 hour before incubation with one of the following primary antibodies: goat polyclonal SDF-1 and fractalkine (1:100; Santa Cruz Biotechnology); goat polyclonal CXCR4, CCR2, and CCR5; rabbit polyclonal CX3CR1 (1:100); mouse monoclonal OX-42 (1:100), GFAP (1:800), CD11b, and CD45 (1:100). Subsequently, sections and coverslips were incubated in the respective rabbit anti-goat, goat anti-rabbit, or horse anti-mouse secondary antibodies for 1 hour, and the reaction products were visualized by treatment with Vectastain ABC kit (Vector Laboratories; Burlingame, CA; http://www.vectorlabs.com) and 3,3-diaminobenzidine tetrahydrochloride (Sigma) as the substrate. Control sections were incubated as above but without the primary antibodies. In addition, immunostaining of CCR5 was performed on parietal cortex, where the expression of CCR5 was shown to be undetectable [24, 25], to confirm the specificity of staining observed in the rMSCs. To serve as positive control, immunostaining of CX3CR1 was also processed on the brainstem sections where the expression of CX3CR1 has been shown in a previous study [26].

Reverse Transcription Polymerase Chain Reaction (RT-PCR) Analysis
Total RNA was isolated from cultured rMSCs and cortical microglia using RNeasy Mini Kit (Qiagen; Chatsworth, CA; http://www.qiagen.com) according to the manufacturer’s instructions and was spectrophotometrically quantified (BioPhotometer; Eppendorf; Hamburg, Germany; http://www.eppendorf.com). The cDNA was prepared from 2.0 µg RNA in the presence of 2.5 µM oligo (dT) primer and 200 U Molony murine leukemia virus reverse transcriptase (M-MLV) (Promega; Madison, WI; http://www.promega.com) in a total volume of 25 µl. The reaction mixture was incubated for 1 hour at 42°C and stopped by heating for 5 minutes at 90°C. Aliquots (1 µl) of cDNA were subsequently amplified (PCR Express; Hybaid, Middlesex, UK; http://www.thermohybaid.com) using specific primers for CCR2, CCR5, CXCR4, and CX3CR1 [21–23], which are listed in Table 1Go. The PCR cycles consist of denaturation at 94°C for 1 minute, annealing at 56°C for 45 seconds, and extension at 72°C for 1 minute, for 40 cycles (CXCR4, CCR5, and CX3CR1); denaturation at 94°C for 1 minute, annealing at 60°C for 45 seconds, and extension at 72°C for 1 minute, for 40 cycles (CCR2). A 10 µl aliquot of each PCR product was size-separated by electrophoresis on a 2% ethidium-bromide-containing agarose gel and photographed. To control for potential genomic DNA contamination, we performed PCR without the reverse transcription step.


View this table:
[in this window]
[in a new window]
  		Table 1. Primers for the RT-PCR analysis
 
Transplantation of rMSCs Into Brain Immediately After Left Hypoglossal Nerve Injury
We used thirty-six male Wistar rats weighing approximately 200 g each in the study on the migration of rMSCs into rat brains after left hypoglossal nerve injury. After anesthetization with an intraperitoneal injection of 1 ml 7% chloral hydrate, left hypoglossal nerves of 24 rats were transected, and the axotomized rats were then transplanted with prelabeled rMSCs. Transplantation surgery was performed in animals mounted on a stereotaxic frame using a Hamilton syringe. Five µl of semisuspended rMSCs (2 x 105 cells/µl) were slowly injecrted into each lateral ventricle (coordinates: 1.0 mm caudal to bregma, ± 1.5 mm lateral to the midline, 4.5 mm from the surface of the cortex) of the rat brain over a period of 5 minutes after left hypoglossal nerve avulsion. The needle was withdrawn 10 minutes later and the scalp sutured. The remaining rats served as untreated controls (n = 4), controls with operation but no injection of rMSCs (n = 4), and controls in which the same number of rMSCs were also injected into the lateral ventricles of sham-operated rats (n = 4). Rats were sacrificed at 1 and 2 weeks after transplantation and perfused with 4% paraformaldehyde in PBS. Serial frozen coronal sections of the brainstem were cut, and every fifth section was viewed under a scanning confocal microscope (LSM 410; Carl Zeiss Microimaging, Inc.; Thornwood, NJ; http://www.zeiss.com). The number of labeled rMSCs in HN was counted and quantitatively analyzed using Student’s t test.

In Vitro Chemotaxis Assay
The in vitro migration of rMSCs in response to recombinant human SDF-1 alpha (rhSDF-1{alpha}) and the extracellular domain of recombinant rat fractalkine (rrfractalkine) (R&D Systems; Minneapolis, MN) was assessed using poly-D-lysine (20 µg/ml, Sigma)-coated polyvinylcarbonate-free membranes (Neuroprobe, Inc.; Gaithersburg, MD; http://www.neuroprobe.com) with 8–12 µm pore size in a modified 48-well microchemotaxis Boyden chamber, as previously described [27]. Briefly, 5 x 106 cells/ml in 50 µl serum-free migration medium Iscove’s modified Dulbecco’s medium (IMDM) + 0.5% BSA was added to the upper chambers. To observe chemotaxis, 25 µl of rhSDF-1{alpha} or rrfractalkine was added to the lower chambers in different concentrations ranging from 0 ng/ml to 500 ng/ml in serum-free IMDM. To observe chemokinesis, rhSDF-1{alpha} and fractalkine were also added to the upper chambers. To determine whether rMSC migration was heterotrimeric G protein-mediated, in these experiments the cells were preincubated with 100 ng/ml pertussis toxin (PTx, Sigma) for 2 hours at 37°C, washed, and loaded in the upper chambers. In order to rule out the toxic effect of PTx on rMSCs, MTT assay was performed as previously described [28]. Briefly, rMSCs were plated and grown to confluency in a 96-well plate. The cells were treated with different concentrations of PTx (50–500 ng/ml) for 2 hours at 37°C. Subsequently, 10 µl of a 5 mg/ml MTT solution in {alpha}-MEM (minimal essential medium) were added to 100 µl of medium in culture wells and incubated for 4 hours at 37°C. The reaction was stopped by adding 100 µl of 0.33% HCl in isopropanyl alcohol. The plate was maintained overnight at 37°C, and the absorption values at 570 nm and 630 nm were then measured using an automatic microtiter plate reader. The experiments were performed in triplicate, and cell viability was expressed as percentage of untreated samples.

In experiments to test the specificity of the chemotactic effect of rhSDF-1{alpha} and rrfractalkine, anti-SDF-1 or anti-fractalkine polyclonal antibodies (Santa Cruz Biotechology) were loaded at the concentration of 480 µg/ml along with rhSDF-1{alpha} or rrfractalkine in the lower chambers, respectively. After 6 hours of incubation at 37°C in 5% CO2, the upper surface of the membrane were scraped free of cells and debris. The membrane was then fixed and stained using hematoxylin staining. Cells that had migrated through pores and adhered to the lower surface of the membrane were analyzed under high-power (x 400) light microscopy and counted in five random high-power fields. Experiments were performed in triplicate, and data were expressed as means of numbers of cells per high-power field (cells/HPF) ± standard error (SE). Data were analyzed for statistical significance between groups using Student’s t test.

In Vivo rMSCs Chemotaxis Assay
Immediately following jugular vein injection of CFDA-SE-labeled rMSCs (3 x 106 cells/rat), the Wistar rat received an intracerebral injection of rhSDF-1{alpha} (0.1 µg in 10 µl) into the right cerebral cortex (coordinates: 1.0 mm caudal to bregma, 1.5 mm lateral to the midline, 2 mm from the surface of the cortex). As control, the opposite side was injected with PBS alone. Approximately 1 cm3 of cortical tissue block around the injected site was excised 6 hours later and proteolytically digested to produce a single-cell suspension. The number of labeled rMSCs per biopsy sample was estimated by flow cytometry. This experiment was repeated in triplicate, and data were expressed as percent of migrated rMSCs of total injected cells ± SE. Data were analyzed for statistical significance using Student’s t test.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In Vitro Characterization of rMSCs
MSCs were isolated from the femurs and tibiae of adult rats and propagated in vitro. As reported in a previous study [19], rMSCs grew as colonies (Fig. 1AGo). For transplantation studies, the rMSCs labeled by CFDA-SE displayed green fluorescence (Fig. 1BGo). There was no evidence of hematopoietic precursors in the cultures as fluorescent cell sorting and immunohistochemistry analysis (data not shown) demonstrated that the cells were negative for CD11b and CD45, cell-surface markers associated with lymphohematopoietic cells.



View larger version (122K):
[in this window]
[in a new window]
  		Figure 1. Characterization of rMSCs. A) Phase-contrast microphotography of rMSCs at passage 0. B) rMSCs displayed green fluorescence after being labeled with CFDA-SE before transplantation. C) Differentiation of rMSCs into osteoblasts in vitro; rMSCs incubated in osteogenic medium for 21 days. The mineral (arrows) in the cultures was detected by staining with Alizarin red. D) Differentiation of rMSCs into adipocytes in vitro; rMSCs incubated in adipogenic medium for 21 days showed fat droplets (arrows) in the cells stained with Oil Red O. Scale bar = 100 µm.

 
To assess the multipotentiality of rMSCs in the culture, the cells were subjected to in vitro differentiation assays. Under the influence of osteogenic medium, the isolated rMSCs formed aggregation or nodules and showed increased calcium accumulation as revealed by Alizarin red staining (Fig. 1CGo). In addition, rMSCs differentiated into adipocytes that were characterized by round morphology and accumulation of large cytoplasmic vacuoles containing lipid as shown by Oil Red O staining (Fig. 1DGo). Collectively, these results demonstrate that rMSCs in the cultures are morphologically and functionally characteristic of multipotential mesenchymal progenitors.

Expression of Chemokine Receptors CCR2, CCR5, CXCR4, and CX3CR1 in rMSCs
We examined the expression of chemokine receptors CCR2, CCR5, CXCR4, and CX3CR1 in rMSCs by RT-PCR, flow cytometry, and immunohistochemistry. The purity of microglial cultures was found to be around 95% (data not shown). We detected mRNA expression of CXCR4, CCR2, CCR5, and CX3CR1 in both microglial cells (positive control) (Fig. 2Aa-dGo, lane 2) and rMSCs (Fig. 2Aa-dGo, lane 3). The protein expression of CCR2, CCR5, CXCR4, and CX3CR1 on the surface of rMSCs was detected by flow cytometry (Fig. 2Ba-dGo). Immunocytochemical analysis revealed the localization of CCR2, CCR5, CXCR4, and CX3CR1 expression on the membranes and in the cytoplasm (Fig. 2Ca-d).



View larger version (49K):
[in this window]
[in a new window]
  		Figure 2. Expression of CCR2, CCR5, CXCR4, and CX3CR1 by rMSCs at mRNA and protein levels. A) RT-PCR was used to determine mRNA expression of chemokine receptors by rMSCs. The PCR bands for CCR2, CCR5, CXCR4, and CX3CR1 were present in both microglial positive control (a-d, lane 2) and rMSCs (a-d, lane 3), whereas they were absent in negative controls (a-d, lane 4) where PCR was performed on RNA template without reverse transcription step. B) Expression of CCR2, CCR5, CXCR4, and CX3CR1 at the protein level in rMSCs; rMSCs were stained with anti-CCR2 (a), CCR5 (b), CXCR4 (c), or CX3CR1 (d) antibodies and then analyzed by flow cytometry. The shaded regions represent isotype control staining and the open regions represent the chemokine receptors staining. C) Localization of CCR2, CCR5, CXCR4, and CX3CR1 in rMSCs. Immunohistochemistry revealed the localization of CCR2 (a), CCR5 (b), CXCR4 (c), and CX3CR1 (d) in the membrane and cytoplasm of rMSCs. Scale bar = 100 µm.

 
Migration of rMSCs in Response to rrfractalkine and rhSDF-1{alpha} in Heterotrimeric G-Protein-Dependent Manner In Vitro
In view of the observations that rMSCs express CXCR4 (the sole receptor for SDF-1) and CX3CR1 (the only receptor for fractalkine), we used rhSDF-1{alpha} and the extracellular domain of rrfractalkine in an in vitro chemotaxis assay. The rrfractalkine (5 ng/ml and 50 ng/ml) and rhSDF-1{alpha} (5–500 ng/ml) significantly (p < 0.01) induced the migration of rMSCs (Figs. 3Go and 4AGo). Interestingly, at higher concentrations of rrfractalkine (250 ng/ml and 500 ng/ml) there was no significant number of migrated rMSCs compared with the control (Fig. 3AGo). However, rhSDF-1{alpha} induced the migration of rMSCs in a dose-dependent manner; the optimal migration of rMSCs was observed at 250 ng/ml of rhSDF-1{alpha} (Fig. 4AGo). The number of migrated rMSCs to rrfractalkine and rhSDF-1{alpha} was decreased when the same effective concentrations of rrfractalkine and rhSDF-1{alpha} were added into both the top and bottom chambers in the assays (Figs. 3Go and 4BGo). Moreover, the chemotactic effects of rrfractalkine or rhSDF-1{alpha} on rMSCs were abolished by the addition of antifractalkine or anti-SDF-1 respectively in the bottom chamber (Figs. 3Go and 4CGo), suggesting the specificity of their chemotactic effects.



View larger version (22K):
[in this window]
[in a new window]
  		Figure 3. Effect of rrfractalkine on migration of rMSCs. A) A significant increase in the number of migrated rMSCs was found at the concentrations of 5 and 50 ng/ml rrfractalkine (p < 0.01) compared with control. After treatment with PTx, the number of migrated rMSCs decreased to the control level at the concentrations of 5 and 50 ng/ml rrfractalkine. B) Chemotactic effect of rrfractalkine on migration of rMSCs. A significant increase in the number of migrated rMSCs was observed at the concentration of 5 ng/ml rrfractalkine. After 5 ng/ml rrfractalkine was added to both top and bottom chambers, the number of migrated rMSCs was significantly decreased. C) Specificity of the effect of rrfractalkine on migration of rMSCs. After blockage of rrfractalkine by 480 µg/ml antifractalkine antibody, the chemotactic effect of rrfractalkine on rMSCs was abolished. **p < 0.01. The results are the mean ± SE of three independent experiments.

 


View larger version (39K):
[in this window]
[in a new window]
  		Figure 4. Effect of rhSDF-1{alpha} on migration of rMSCs. A) The number of migrated rMSCs increased dose-dependently at the concentration of 5–500 ng/ml (p < 0.01), compared with control. Maximum effect of rhSDF-1{alpha} was observed at the concentration of 250 ng/ml. Pretreatment of rMSCs with PTx abrogated the effect of rhSDF-1{alpha} at various concentrations. a) The migrated rMSCs in the control condition. b) The migrated rMSCs at the concentration of 250 ng/ml rhSDF-1{alpha}. c) The migrated rMSCs at the concentration of 250 ng/ml rhSDF-1{alpha} after pretreatment of PTx. B) Chemotactic effect of rhSDF-1{alpha} on rMSCs. When 250 ng/ml rhSDF-1{alpha} was added to both top and bottom chambers, the number of migrated rMSCs was significantly decreased. a) The migrated rMSCs in the control condition. b) The migrated rMSCs at the concentration of 250 ng/ml rhSDF-1{alpha} in the bottom chamber only. c) The migrated rMSCs at the concentration of 250 ng/ml rhSDF-1{alpha} added to both top and bottom chambers. C) Specificity of the effect of rhSDF-1{alpha} on migration of rMSCs. When rhSDF-1{alpha} was blocked by 480 µg/ml anti-SDF-1 antibody, the chemotactic effect of rhSDF-1{alpha} on rMSCs was abolished. a) The migrated rMSCs in the control condition. b) The migrated rMSCs at the concentration of 250 ng/ml rhSDF-1{alpha}. c) The migrated rMSCs at the concentration of 250 ng/ml rhSDF-1{alpha} blocked by anti-SDF-1 antibody. **p < 0.01. Scale bar = 100 µm. The results are the mean ± SE of three independent experiments.

 
In order to understand whether the rrfractalkine- and rhSDF-1{alpha}-induced migration of rMSCs was transmitted in a G-protein-dependent manner, the chemotaxis assay with the rrfractalkine and rhSDF-1{alpha} was performed in the presence of PTx, a specific inhibitor of heterotrimeric G-protein coupling to G-protein-coupled receptors, potently inhibiting signaling through Gai proteins [29]. Upon pre-exposure of rMSCs to PTx, almost complete inhibition of rrfractalkine- and rhSDF-1{alpha}-induced migratory response was observed (Figs. 3Go and 4AGo). However, PTx was not observed to affect the viability of rMSCs (data not shown). These results indicate that the migratory response of rMSCs induced by rrfractalkine and rhSDF-1{alpha} is mediated by heterotrimeric G proteins, possibly Gai, suggesting that the signaling events leading to migration of rMSCs are transmitted through the CX3CR1 and CXCR4 receptors, both of which are G-protein-coupled receptors.

Migration of rMSCs Following the Intracerebral Injection of rhSDF-1{alpha} In Vivo
Based on the observation that rhSDF-1{alpha} has a potent migratory effect on rMSCs in vitro, we investigated whether rhSDF-1{alpha} injected intracerebrally could promote the migration of tranaplanted rMSCs in vivo. As shown by flow cytometry analysis, administration of rhSDF-1{alpha} (0.1 µg) significantly (p < 0.01) induced the accumulation of prelabeled rMSCs in the cerebral cortex area surrounding the injection site compared with the effect of PBS alone (Fig. 5Go).



View larger version (16K):
[in this window]
[in a new window]
  		Figure 5. Effect of rhSDF-1{alpha} on migration of rMSCs in vivo. Immediately following jugular-vein injection of CFDA-SE–labeled rMSCs (3 x 106 cells/rat), the Wistar rat received an intracerebral injection of rhSDF-1{alpha} (0.1 µg in 10 µl) into the right cerebral cortex. As control, the opposite side was injected with PBS alone. Approximately 1 cm3 of cortical tissue block around the injected site was excised 6 hours later and proteolytically digested to produce a single-cell suspension. The number of labeled rMSCs per biopsy sample was estimated by flow cytometry and expressed as a percentage of the total number of fluorescent rMSCs injected into the jugular vein. The percentage of migrated rMSCs in the rhSDF-1{alpha}-injected sites was significantly higher (p < 0.01) compared with the PBS-injected site. **p < 0.01. The results are the mean ± SE of three independent experiments.

 
Selective Migration of Transplanted rMSCs Into Injured Left HN
To further prove that transplanted rMSCs possess the directed migratory capacity to target impaired sites in the brain, we investigated their trafficking in an animal model of left hypoglossal nerve avulsion. Before transplantation, the cells were prelabeled with CFDA-SE. Examination of frozen sections by fluorescent microscopy revealed that at 1 and 2 weeks after injection of CFDA-SE–labeled MSCs into both lateral ventricles of the rat brain with left hypoglossal nerve avulsion, significantly more (p < 0.01) labeled cells were detected in the injured HN (Fig. 6CGo, 6EGo), in comparison with that of the right control side (Fig. 6DGo, 6FGo) and sham-operated control rats (Fig. 6AGo, 6BGo). Moreover, at 2 weeks after transplantation, the number of rMSCs in the avulsed HN was significantly (p < 0.01) decreased compared with that at 1 week postoperation (Fig. 6GGo). This observation suggests that transplanted MSCs may have the capacity for preferential migration to the site of neuronal injury.



View larger version (68K):
[in this window]
[in a new window]
  		Figure 6. Migration of rMSCs in the model of left hypoglossal nerve avulsion. Left hypoglossal nerves of rats were avulsed and 5 µl of semisuspended prelabeled rMSCs (2 x 105 cells/µl) were then transplanted into the lateral ventricles of the avulsed and sham-operated rats. A few migrated rMSCs (arrows) were observed in the left avulsed HN (C, E), but not in the right HN (D, F) and controls (A, B), at 1 week (C, D) and 2 weeks (E, F) after operation and transplantation of labeled rMSCs into both lateral ventricles. Note: there were no labeled rMSCs in adjacent left dorsal motor nucleus of the vagus (DMV) (E). There were significantly more rMSCs in the left HN (p < 0.01) compared with right HN and control rats at 1 week after transplantation and operation (G). Two weeks later, the number of rMSCs was significantly decreased (p < 0.01) (G). **p < 0.01. Scale bar = 100 µm. The results are the mean ± SE of three independent experiments.

 
Upregulated Expression of Fractalkine and SDF-1 in the Injured HN
We examined the expression of fractalkine and SDF-1 in the HN at 1 and 2 weeks after left hypoglossal nerve avulsion. Immunohistochemical staining showed that, compared with the weak expression of fractalkine and SDF-1 in the HN of control rats (Fig. 7Aa,bGo; 7Ba,bGo) and in the right HN of avulsed rats (Fig. 7AdGo, 7BdGo), immunoreactivities were dramatically upregulated in the left HN at 1 week after the nerve injury (Fig. 7AcGo, 7BcGo). The expression of fractalkine and SDF-1 remained enhanced at 2 weeks after operation (Fig. 7AeGo, 7BeGo). Quantification analysis of the immunopositive cells showed that the numbers of fractalkine and SDF-1 immunopositive cells in the left avulsed HN were significantly (p < 0.01) increased at 1 week after operation compared with that in the right HN and controls (Fig. 7AgGo, 7BgGo). Subsequently, at 2 weeks after operation, the numbers of MCP-1, fractalkine, and SDF-1 positive cells were significantly (p < 0.01) decreased (Fig. 7AgGo, 7BgGo).



View larger version (104K):
[in this window]
[in a new window]
  		Figure 7. Expression of fractalkine and SDF-1 in the HN. A) Fractalkine immunostaining was upregulated in the left HN at 1 (c) and 2 (e) weeks after operation compared with the controls (a, b, d, f). Quantification analysis revealed that the number of fractalkine immuopositive cells significantly (p < 0.01) increased in the left HN at 1 week after operation compared with the controls (g). Subsequently, at 2 weeks after operation, the number significantly decreased (p < 0.01) (g). B) Expression of SDF-1 in the HN. SDF-1 immunoreactivity was enhanced in left HN at 1 week (c) and 2 weeks (e) after operation, compared with the controls (a, b, d, f). Quantification analysis revealed that the number of SDF-1 immunoreactive cells significantly (p < 0.01) increased in the left HN at 1 week after operation compared with the controls (g). Subsequently, at 2 weeks after operation, the number significantly decreased (p < 0.01) (g). **p < 0.01. Scale bar = 100 µm. The results are the mean ± SE.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The directed migration of rMSCs transplanted intravenously, intra-arterially, or intracerebrally into injured or diseased sites in the brain recently has been reported in animal models of brain trauma and ischemia [11–15]. Our study is the first report showing the migration of rMSCs transplanted into the cerebrospinal fluid into the injured nucleus in the brain of a remote nerve injury animal model. It has been shown that the chemotactic proteins MCP-1, MIP-1{alpha}, and interleukin-8 promote the migration of human MSCs (hMSCs) in interface cultures [16]. In this study, we have demonstrated that the interaction of chemokines with their receptors could mediate the trafficking of rMSCs to the impaired site in the brain.

Chemokines constitute a family of small secreted proteins (8–13 kDa) that are best known for their ability to cause activation and migration of leukocytes [30–32]. Based on the relative position of the cysteine residues in the protein sequence, they are classified into four subfamilies: CXC ({alpha}) and CC (ß) chemokines, fractalkine (CXXXC), a membrane-bound glycoprotein, and lymphotactin [30, 33–35]. The {alpha}- and ß-chemokines and fractalkine exert their effects through the CXC receptors (CXCR), CC receptors (CCR), and CX3CR1 receptor, respectively. In our study, the chemokine receptors CXCR4, CCR2, CCR5, and CX3CR1 were found to be expressed in rMSCs at mRNA and protein levels.

It has been shown that specific interaction between chemokines and their receptors mediates the migratory process of leukocytes including neutrophils, monocytes, and T cells into inflammatory sites [36]. The migration of rMSCs in response to fractalkine and SDF-1{alpha} observed in our study indicates that interaction between chemokines and their receptors could be involved in the preferential migration of rMSCs. Interestingly, SDF-1{alpha} appears to be the more potent chemotactic factor for the migration of rMSCs as it induced significantly more rMSCs to migrate than did fractalkine.

Chemokine-induced activation of chemokine receptors has been shown to be mediated primarily by members of the Gai subclass of G proteins and also by other members of the Ga class [37–40]. In this study, the fractalkine- and SDF-1{alpha}-induced migration of rMSCs in vitro was interfered with by PTx, a specific inhibitor of Gai protein, indicating that the migratory process is possibly mediated by Gai protein. These results further suggest that the signaling events leading to migration of rMSCs could be transmitted through the CX3CR1 and CXCR4 receptors, both being the G-protein-coupled receptors.

SDF-1, as a major constituent of the bone marrow, has been reported to be constitutively expressed in a wide range of tissues including those of the brain, heart, kidney, liver, lung, and spleen [41]. SDF-1 has been shown to participate in hematopoiegensis. Increasing evidence implicates the significant role of SDF-1 and its sole receptor, CXCR4, in the inflammatory processes in several diseases such as allergic airway diseases, rheumatoid arthritis, atherosclerosis, and several brain diseases [42]. Our study reveals the upregulated expression of SDF-1 in the HN after axotomy, suggesting that SDF-1 could be involved in the inflammatory processes in the HN after avulsion.

The interaction of SDF-1 with CXCR4 mediates the homing of hematopoietic stem cells to the bone marrow [43]. SDF-1 has also been reported to induce migration of endothelial and neuronal cells [44, 45]. Recent evidence even suggested a role for SDF-1 and CXCR4 in the development of bone marrow metastases in neuroblastoma [46]. In our study, we demonstrated that SDF-1 induced the migration of rMSCs in vitro and in vivo. Moreover, the increased temporal expression of SDF-1 paralleled the number of migrated CXCR4-expressing rMSCs in avulsed HN. Recently it has been demonstrated that SDF-1 is sufficient to induce the homing of bone-marrow-derived stem cells to injured myocardium in a rat model of myocardial infarction [47]. Hence, it is suggested that the chemotactic interaction of CXCR4-SDF-1 could facilitate the homing of rMSCs to the impaired site in the brain.

Fractalkine is constitutively expressed in a number of nonhematopoietic tissues, including the brain. In the rat CNS, while fractalkine is mainly expressed in neurons and to a lesser extent in astrocytes, its sole receptor, CX3CR1, is specifically expressed on microglia [48, 49]. It exists as a membrane-bound protein containing a chemokine domain tethered on a long, mucin-like stalk. Its extracellular domain can potentially be released as a soluble protein by proteolysis at the conserved dibasic motif proximal to the transmembrane region [33, 50]. Interacting with CX3CR1, membrane-bound fractalkine promotes adhesion of leukocytes while soluble protein of human fractalkine is chemotactic for T cells and monocytes. In mice, the soluble form of fractalkine induces the chemotaxis of neutrophils and T lymphocytes [33, 50, 51]. Moreover, it has been shown that the soluble form of rat fractalkine chemoattracts microglia [48]. Our study provides the first evidence for the expression of CX3CR1 at both mRNA and protein levels on rMSCs and the chemotaxis of rMSCs in response to the soluble form of rat fractalkine in vitro.

In the model of facial nerve transection, the increased soluble form of fractalkine has been shown to mediate the chemotaxis of microglia in the lesioned facial motor nucleus [48]. In our study, the increased immunoreactivity of fractalkine correlates with the number of migrated CX3CR1-expressing rMSCs in the HN after avulsion, which, along with the chemotactic effect of the soluble form of rat fractalkine on rMSCs in vitro, suggests a possible role for the soluble form of fractalkine and CX3CR1 complex in the preferential migration of transplanted rMSCs.

It has been shown that MCP-1 promotes the migration of rMSCs that express CCR2 [17]. We confirmed the expression of CCR2 at both mRNA and protein levels in the rMSCs. MIP-1{alpha}, which has been shown to promote the migration of human MSCs in interface cultures [16], may also have the potential to induce the migration of rMSCs, as the expression of its receptor, CCR5, was detected in the rMSCs.

In conclusion, results from this study suggest that the interaction of fractalkine-CX3CR1 and SDF-1–CXCR4 could play important roles in the directed migration of transplanted rMSCs to impaired sites in the brain. However, it is not clear whether endogenous rMSCs could home to the fractalkine- or SDF-1–expressing sites in various tissues of the body in physiological and pathological conditions. Further investigation may be necessary to understand the possible roles of endogenous MSCs in participating in organogenesis in normal conditions or in the repair processes in pathological conditions.

It has been shown that rMSCs could differentiate into neurons and astrocytes in vivo, indicating that MSCs are potentially useful as vectors for treating a variety of CNS disorders [52, 53]. Although recent evidence raised doubt on the issue of transdifferentiation [54, 55], rMSCs have been shown to produce growth factors and neurotrophins that may improve the functions of the impaired brain [56–58]. Therefore, illustration of molecular signals mediating the trafficking of rMSCs could contribute to the development of rMSC-mediated cell therapy strategies in terms of facilitating site-specific migration of transplanted stem cells, thereby promoting functional improvement of the diseased or injured brain.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This work was supported by a research grant (R181-000-059-213) from the National University of Singapore.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Piersma AH, Brockbank KG, Ploemacher RE et al. Characterization of fibroblastic stromal cells from murine bone marrow. Exp Hematol 1985;13:237–243.[Medline]

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

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

  4. Clark BR, Keating A. Biology of bone marrow stroma. Ann N Y Acad Sci 1995;770:70–78.[Abstract]

  5. 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 USA 1998;95:1142–1147.[Abstract/Free Full Text]

  6. Wakitani S, Saito T, Caplan AI. Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle Nerve 1995;18:1417–1426.[CrossRef][Medline]

  7. Azizi SA, Stokes D, Augelli BJ et al. Engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats—similarities to astrocyte grafts. Proc Natl Acad Sci USA 1998;95:3908–3913.[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. Woodbury D, Schwarz EJ, Prockop DJ et al. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res 2000;61:364–370.[CrossRef][Medline]

  10. Prockop DJ, Azizi SA, Phinney DG et al. Potential use of marrow stromal cells as therapeutic vectors for diseases of the central nervous system. Prog Brain Res 2000;128:293–297.[Medline]

  11. Lu D, Li Y, Wang L et al. Intraarterial administration of marrow stromal cells in a rat model of traumatic brain injury. J Neurotrauma 2001;18:813–819.[CrossRef][Medline]

  12. Lu D, Mahmood A, Wang L et al. Adult bone marrow stromal cells administered intravenously to rats after traumatic brain injury migrate into brain and improve neurological outcome. Neuroreport 2001;12:559–563.[CrossRef][Medline]

  13. Mahmood A, Lu D, Wang L et al. Treatment of traumatic brain injury in female rats with intravenous administration of bone marrow stromal cells. Neurosurgery 2001;49:1196–1203.[Medline]

  14. Chen J, Li Y, Wang L et al. Therapeutic benefit of intracerebral transplantation of bone marrow stromal cells after cerebral ischemia in rats. J Neurol Sci 2001;189:49–57.[CrossRef][Medline]

  15. Chen J, Li Y, Wang L et al. Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats. Stroke 2001;32:1005–1011.[Abstract/Free Full Text]

  16. Wang L, Li Y, Chen X et al. MCP-1, MIP-1, IL-8 and ischemic cerebral tissue enhance human bone marrow stromal cell migration in interface culture. Hematology 2002;7:113–117.[CrossRef][Medline]

  17. Wang L, Li Y, Chen J et al. Ischemic cerebral tissue and MCP-1 enhance rat bone marrow stromal cell migration in interface culture. Exp Hematol 2002;30:831–836.[CrossRef][Medline]

  18. Jiang Y, Zhang M, Koishi K et al. TGF-beta 2 attenuates the injury-induced death of mature motoneurons. J Neurosci Res 2000;62:809–813.[CrossRef][Medline]

  19. Javazon EH, Colter DC, Schwarz EJ et al. Rat marrow stromal cells are more sensitive to plating density and expand more rapidly from single-cell-derived colonies than human marrow stromal cells. STEM CELLS 2001;19:219–225.[Abstract/Free Full Text]

  20. Digirolamo CM, Stokes D, Colter D et al. Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate. Br J Haematol 1999;107:275–281.[CrossRef][Medline]

  21. Boddeke EW, Meigel I, Frentzel S et al. Cultured rat microglia express functional beta-chemokine receptors. J Neuroimmunol 1999;98:176–184.[CrossRef][Medline]

  22. Boddeke EW, Meigel I, Frentzel S et al. Functional expression of the fractalkine (CX3C) receptor and its regulation by lipopolysaccharide in rat microglia. Eur J Pharmacol 1999;374:309–313.[CrossRef][Medline]

  23. Ohtani Y, Minami M, Kawaguchi N et al. Expression of stromal cell-derived factor-1 and CXCR4 chemokine receptor mRNAs in cultured rat glial and neuronal cells. Neurosci Lett 1998;249:163–166.[CrossRef][Medline]

  24. Bonavia R, Bajetto A, Barbero S et al. Chemokines and their receptors in the CNS: expression of CXCL12/SDF-1 and CXCR4 and their role in astrocyte proliferation. Toxicol Lett 2003;139:181–189.[CrossRef][Medline]

  25. Bajetto A, Bonavia R, Barbero S et al. Expression of chemokine receptors in the rat brain. Ann N Y Acad Sci 1999;876:201–209.[Abstract/Free Full Text]

  26. Nishiyori A, Minami M, Ohtani Y et al. Localization of fractalkine and CX3CR1 mRNAs in rat brain: does fractalkine play a role in signaling from neuron to microglia? FEBS Lett 1998;429:167–172.[CrossRef][Medline]

  27. Garcia-Zepeda EA, Rothenberg ME, Ownbey RT et al. Human eotaxin is a specific chemoattractant for eosinophil cells and provides a new mechanism to explain tissue eosinophilia. Nat Med 1996;2:449–456.[CrossRef][Medline]

  28. Korting HC, Schindler S, Hartinger A et al. MTT-assay and neutral red release (NRR)-assay: relative role in the prediction of the irritancy potential of surfactants. Life Sci 1994;55:533–540.[CrossRef][Medline]

  29. Fields TA, Casey PJ. Signalling functions and biochemical properties of pertussis toxin-resistant G-proteins. Biochem J 1997;321:561–571.

  30. Baggiolini M. Chemokines and leukocyte traffic. Nature 1998;392:565–568.[CrossRef][Medline]

  31. Melchers F, Rolink AG, Schaniel C. The role of chemokines in regulating cell migration during humoral immune responses. Cell 1999;99:351–354.[CrossRef][Medline]

  32. Murdoch C, Finn A. Chemokine receptors and their role in inflammation and infectious diseases. Blood 2000;95:3032–3043.[Abstract/Free Full Text]

  33. Bazan JF, Bacon KB, Hardiman G et al. A new class of membrane-bound chemokine with a CX3C motif. Nature 1997;385:640–644.[CrossRef][Medline]

  34. Kelner GS, Kennedy J, Bacon KB et al. Lymphotactin: a cytokine that represents a new class of chemokine. Science 1994;266:1395–1399.[Abstract/Free Full Text]

  35. Zlotnik A, Yoshie O. Chemokines: a new classification system and their role in immunity. Immunity 2000;12:121–127.[CrossRef][Medline]

  36. Luster AD. Chemokines—chemotactic cytokines that mediate inflammation. N Engl J Med 1998;338:436–445.[Free Full Text]

  37. Wu D, LaRosa GJ, Simon MI. G protein-coupled signal transduction pathways for interleukin-8. Science 1993;261:101–103.[Abstract/Free Full Text]

  38. al Aoukaty A, Schall TJ, Maghazachi AA. Differential coupling of CC chemokine receptors to multiple heterotrimeric G proteins in human interleukin-2-activated natural killer cells. Blood 1996;87:4255–4260.[Abstract/Free Full Text]

  39. Arai H, Charo IF. Differential regulation of G-protein-mediated signaling by chemokine receptors. J Biol Chem 1996;271:21814–21819.[Abstract/Free Full Text]

  40. Hesselgesser J, Liang M, Hoxie J et al. Identification and characterization of the CXCR4 chemokine receptor in human T cell lines: ligand binding, biological activity, and HIV-1 infectivity. J Immunol 1998;160:877–883.[Abstract/Free Full Text]

  41. Ma Q, Jones D, Borghesani PR et al. Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice. Proc Natl Acad Sci USA 1998;95:9448–9453.[Abstract/Free Full Text]

  42. Lazarini F, Tham TN, Casanova P et al. Role of the alpha-chemokine stromal cell-derived factor (SDF-1) in the developing and mature central nervous system. Glia 2003;42:139–148.[CrossRef][Medline]

  43. 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]

  44. Hesselgesser J, Halks-Miller M, DelVecchio V et al. CD4-independent association between HIV-1 gp120 and CXCR4: functional chemokine receptors are expressed in human neurons. Curr Biol 1997;7:112–121.[CrossRef][Medline]

  45. Feil C, Augustin HG. Endothelial cells differentially express functional CXC-chemokine receptor-4 (CXCR-4/fusin) under the control of autocrine activity and exogenous cytokines. Biochem Biophys Res Commun 1998;247:38–45.[CrossRef][Medline]

  46. Geminder H, Sagi-Assif O, Goldberg L et al. A possible role for CXCR4 and its ligand, the CXC chemokine stromal cell-derived factor-1, in the development of bone marrow metastases in neuroblastoma. J Immunol 2001;167:4747–4757.[Abstract/Free Full Text]

  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. Harrison JK, Jiang Y, Chen S et al. Role for neuronally derived fractalkine in mediating interactions between neurons and CX3CR1-expressing microglia. Proc Natl Acad Sci USA 1998;95:10896–10901.[Abstract/Free Full Text]

  49. Maciejewski-Lenoir D, Chen SZ, Feng LL et al. Characterization of fractalkine in rat brain cells: migratory and activation signals for CX3CR-1-expressing microglia. J Immunol 1999;163:1628–1635.[Abstract/Free Full Text]

  50. Pan Y, Lloyd C, Zhou H et al. Neurotactin, a membrane-anchored chemokine upregulated in brain inflammation. Nature 1997;387:611–617.[CrossRef][Medline]

  51. Imai T, Hieshima K, Haskell C et al. Identification and molecular characterization of fractalkine receptor CX3CR1, which mediates both leukocyte migration and adhesion. Cell 1997;91:521–530.[CrossRef][Medline]

  52. Sanchez-Ramos JR. Neural cells derived from adult bone marrow and umbilical cord blood. J Neurosci Res 2002;69:880–893.[CrossRef][Medline]

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

  54. Ying QL, Nichols J, Evans EP et al. Changing potency by spontaneous fusion. Nature 2002;416:545–548.[CrossRef][Medline]

  55. Terada N, Hamazaki T, Oka M et al. Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature 2002;416:542–545.[CrossRef][Medline]

  56. Chen X, Li Y, Wang L et al. Ischemic rat brain extracts induce human marrow stromal cell growth factor production. Neuropathology 2002;22:275–279.[CrossRef][Medline]

  57. Chen X, Katakowski M, Li Y et al. Human bone marrow stromal cell cultures conditioned by traumatic brain tissue extracts: growth factor production. J Neurosci Res 2002;69:687–691.[CrossRef][Medline]

  58. Li Y, Chen J, Chen XG et al. Human marrow stromal cell therapy for stroke in rat: neurotrophins and functional recovery. Neurology 2002;59:514–523.[Abstract/Free Full Text]

Received September 17, 2003; accepted for publication December 15, 2003.



This article has been cited by other articles:


Home page
 Stem CellsHome page
S. Otsuru, K. Tamai, T. Yamazaki, H. Yoshikawa, and Y. Kaneda
Circulating Bone Marrow-Derived Osteoblast Progenitor Cells Are Recruited to the Bone-Forming Site by the CXCR4/Stromal Cell-Derived Factor-1 Pathway
Stem Cells, January 1, 2008; 26(1): 223 - 234.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page
X. Cui, J. Chen, A. Zacharek, Y. Li, C. Roberts, A. Kapke, S. Savant-Bhonsale, and M. Chopp
Nitric Oxide Donor Upregulation of Stromal Cell-Derived Factor-1/Chemokine (CXC Motif) Receptor 4 Enhances Bone Marrow Stromal Cell Migration into Ischemic Brain After Stroke
Stem Cells, November 1, 2007; 25(11): 2777 - 2785.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
M. Zhang, N. Mal, M. Kiedrowski, M. Chacko, A. T. Askari, Z. B. Popovic, O. N. Koc, and M. S. Penn
SDF-1 expression by mesenchymal stem cells results in trophic support of cardiac myocytes after myocardial infarction
FASEB J, October 1, 2007; 21(12): 3197 - 3207.
[Abstract] [Full Text] [PDF]

Home page
 RadiologyHome page
R. Schafer, R. Kehlbach, J. Wiskirchen, R. Bantleon, J. Pintaske, B. R. Brehm, A. Gerber, H. Wolburg, C. D. Claussen, and H. Northoff
Transferrin Receptor Upregulation: In Vitro Labeling of Rat Mesenchymal Stem Cells with Superparamagnetic Iron Oxide
Radiology, August 1, 2007; 244(2): 514 - 523.
[Abstract] [Full Text] [PDF]

Home page
 haematolHome page
M. Shi, J. Li, L. Liao, B. Chen, B. Li, L. Chen, H. Jia, and R. C. Zhao
Regulation of CXCR4 expression in human mesenchymal stem cells by cytokine treatment: role in homing efficiency in NOD/SCID mice
Haematologica, July 1, 2007; 92(7): 897 - 904.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page