Interactions of Chemokines and Chemokine Receptors Mediate the Migration of Mesenchymal Stem Cells to the Impaired Site in the Brain After H
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《干细胞学杂志》
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@nus.edu.sg
ABSTRACT
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 . 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 . 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 .
MSCs have been shown to have multipotential capacities to differentiate into osteoblasts, adipocytes, chondrocytes, myoblasts, and myotubes . Recent data have pointed to the unexpected ability of both human and rodent MSCs to differentiate into neural cells . The evidence has suggested their therapeutic potential to regenerate neural cells in the injured or diseased brain .
Interestingly, recent studies from Chopp’s group 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 . 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 , 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 . 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
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 , rMSCs grew as colonies (Fig. 1A). For transplantation studies, the rMSCs labeled by CFDA-SE displayed green fluorescence (Fig. 1B). 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.
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. 1C). 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. 1D). 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-d, lane 2) and rMSCs (Fig. 2Aa-d, lane 3). The protein expression of CCR2, CCR5, CXCR4, and CX3CR1 on the surface of rMSCs was detected by flow cytometry (Fig. 2Ba-d). Immunocytochemical analysis revealed the localization of CCR2, CCR5, CXCR4, and CX3CR1 expression on the membranes and in the cytoplasm (Fig. 2Ca-d).
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 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 and the extracellular domain of rrfractalkine in an in vitro chemotaxis assay. The rrfractalkine (5 ng/ml and 50 ng/ml) and rhSDF-1 (5–500 ng/ml) significantly (p < 0.01) induced the migration of rMSCs (Figs. 3 and 4A). 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. 3A). However, rhSDF-1 induced the migration of rMSCs in a dose-dependent manner; the optimal migration of rMSCs was observed at 250 ng/ml of rhSDF-1 (Fig. 4A). The number of migrated rMSCs to rrfractalkine and rhSDF-1 was decreased when the same effective concentrations of rrfractalkine and rhSDF-1 were added into both the top and bottom chambers in the assays (Figs. 3 and 4B). Moreover, the chemotactic effects of rrfractalkine or rhSDF-1 on rMSCs were abolished by the addition of antifractalkine or anti-SDF-1 respectively in the bottom chamber (Figs. 3 and 4C), suggesting the specificity of their chemotactic effects.
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.
Figure 4. Effect of rhSDF-1 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 was observed at the concentration of 250 ng/ml. Pretreatment of rMSCs with PTx abrogated the effect of rhSDF-1 at various concentrations. a) The migrated rMSCs in the control condition. b) The migrated rMSCs at the concentration of 250 ng/ml rhSDF-1. c) The migrated rMSCs at the concentration of 250 ng/ml rhSDF-1 after pretreatment of PTx. B) Chemotactic effect of rhSDF-1 on rMSCs. When 250 ng/ml rhSDF-1 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 in the bottom chamber only. c) The migrated rMSCs at the concentration of 250 ng/ml rhSDF-1 added to both top and bottom chambers. C) Specificity of the effect of rhSDF-1 on migration of rMSCs. When rhSDF-1 was blocked by 480 μg/ml anti-SDF-1 antibody, the chemotactic effect of rhSDF-1 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. c) The migrated rMSCs at the concentration of 250 ng/ml rhSDF-1 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-induced migration of rMSCs was transmitted in a G-protein-dependent manner, the chemotaxis assay with the rrfractalkine and rhSDF-1 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 . Upon pre-exposure of rMSCs to PTx, almost complete inhibition of rrfractalkine- and rhSDF-1-induced migratory response was observed (Figs. 3 and 4A). 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 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 In Vivo
Based on the observation that rhSDF-1 has a potent migratory effect on rMSCs in vitro, we investigated whether rhSDF-1 injected intracerebrally could promote the migration of tranaplanted rMSCs in vivo. As shown by flow cytometry analysis, administration of rhSDF-1 (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. 5).
Figure 5. Effect of rhSDF-1 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 (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-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. 6C, 6E), in comparison with that of the right control side (Fig. 6D, 6F) and sham-operated control rats (Fig. 6A, 6B). 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. 6G). This observation suggests that transplanted MSCs may have the capacity for preferential migration to the site of neuronal injury.
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,b; 7Ba,b) and in the right HN of avulsed rats (Fig. 7Ad, 7Bd), immunoreactivities were dramatically upregulated in the left HN at 1 week after the nerve injury (Fig. 7Ac, 7Bc). The expression of fractalkine and SDF-1 remained enhanced at 2 weeks after operation (Fig. 7Ae, 7Be). 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. 7Ag, 7Bg). Subsequently, at 2 weeks after operation, the numbers of MCP-1, fractalkine, and SDF-1 positive cells were significantly (p < 0.01) decreased (Fig. 7Ag, 7Bg).
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
This work was supported by a research grant (R181-000-059-213) from the National University of Singapore.
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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@nus.edu.sg
ABSTRACT
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 . 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 . 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 .
MSCs have been shown to have multipotential capacities to differentiate into osteoblasts, adipocytes, chondrocytes, myoblasts, and myotubes . Recent data have pointed to the unexpected ability of both human and rodent MSCs to differentiate into neural cells . The evidence has suggested their therapeutic potential to regenerate neural cells in the injured or diseased brain .
Interestingly, recent studies from Chopp’s group 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 . 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 , 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 . 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
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 , rMSCs grew as colonies (Fig. 1A). For transplantation studies, the rMSCs labeled by CFDA-SE displayed green fluorescence (Fig. 1B). 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.
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. 1C). 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. 1D). 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-d, lane 2) and rMSCs (Fig. 2Aa-d, lane 3). The protein expression of CCR2, CCR5, CXCR4, and CX3CR1 on the surface of rMSCs was detected by flow cytometry (Fig. 2Ba-d). Immunocytochemical analysis revealed the localization of CCR2, CCR5, CXCR4, and CX3CR1 expression on the membranes and in the cytoplasm (Fig. 2Ca-d).
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 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 and the extracellular domain of rrfractalkine in an in vitro chemotaxis assay. The rrfractalkine (5 ng/ml and 50 ng/ml) and rhSDF-1 (5–500 ng/ml) significantly (p < 0.01) induced the migration of rMSCs (Figs. 3 and 4A). 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. 3A). However, rhSDF-1 induced the migration of rMSCs in a dose-dependent manner; the optimal migration of rMSCs was observed at 250 ng/ml of rhSDF-1 (Fig. 4A). The number of migrated rMSCs to rrfractalkine and rhSDF-1 was decreased when the same effective concentrations of rrfractalkine and rhSDF-1 were added into both the top and bottom chambers in the assays (Figs. 3 and 4B). Moreover, the chemotactic effects of rrfractalkine or rhSDF-1 on rMSCs were abolished by the addition of antifractalkine or anti-SDF-1 respectively in the bottom chamber (Figs. 3 and 4C), suggesting the specificity of their chemotactic effects.
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.
Figure 4. Effect of rhSDF-1 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 was observed at the concentration of 250 ng/ml. Pretreatment of rMSCs with PTx abrogated the effect of rhSDF-1 at various concentrations. a) The migrated rMSCs in the control condition. b) The migrated rMSCs at the concentration of 250 ng/ml rhSDF-1. c) The migrated rMSCs at the concentration of 250 ng/ml rhSDF-1 after pretreatment of PTx. B) Chemotactic effect of rhSDF-1 on rMSCs. When 250 ng/ml rhSDF-1 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 in the bottom chamber only. c) The migrated rMSCs at the concentration of 250 ng/ml rhSDF-1 added to both top and bottom chambers. C) Specificity of the effect of rhSDF-1 on migration of rMSCs. When rhSDF-1 was blocked by 480 μg/ml anti-SDF-1 antibody, the chemotactic effect of rhSDF-1 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. c) The migrated rMSCs at the concentration of 250 ng/ml rhSDF-1 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-induced migration of rMSCs was transmitted in a G-protein-dependent manner, the chemotaxis assay with the rrfractalkine and rhSDF-1 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 . Upon pre-exposure of rMSCs to PTx, almost complete inhibition of rrfractalkine- and rhSDF-1-induced migratory response was observed (Figs. 3 and 4A). 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 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 In Vivo
Based on the observation that rhSDF-1 has a potent migratory effect on rMSCs in vitro, we investigated whether rhSDF-1 injected intracerebrally could promote the migration of tranaplanted rMSCs in vivo. As shown by flow cytometry analysis, administration of rhSDF-1 (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. 5).
Figure 5. Effect of rhSDF-1 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 (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-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. 6C, 6E), in comparison with that of the right control side (Fig. 6D, 6F) and sham-operated control rats (Fig. 6A, 6B). 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. 6G). This observation suggests that transplanted MSCs may have the capacity for preferential migration to the site of neuronal injury.
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,b; 7Ba,b) and in the right HN of avulsed rats (Fig. 7Ad, 7Bd), immunoreactivities were dramatically upregulated in the left HN at 1 week after the nerve injury (Fig. 7Ac, 7Bc). The expression of fractalkine and SDF-1 remained enhanced at 2 weeks after operation (Fig. 7Ae, 7Be). 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. 7Ag, 7Bg). Subsequently, at 2 weeks after operation, the numbers of MCP-1, fractalkine, and SDF-1 positive cells were significantly (p < 0.01) decreased (Fig. 7Ag, 7Bg).
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
This work was supported by a research grant (R181-000-059-213) from the National University of Singapore.
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