Published online before print August 28, 2008, doi: 10.1161/STROKEAHA.108.514612
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(Stroke. 2008;39:3411.)
© 2008 American Heart Association, Inc.
Original Contributions |
Antagonism of Sphingosine 1-Phosphate Receptor-2 Enhances Migration of Neural Progenitor Cells Toward an Area of Brain Infarction
From the Department of Orthopedic Surgery (A.K., Y.H.), the Center for Molecular Medicine (T.O., Y.K., S.M., J.M., Y.S.), and the Department of Physiology (K.S.), Jichi Medical University School of Medicine, Tochigi, and the Department of Clinical Laboratory Medicine (R.O., Y.Y.), Graduate School of Medicine, University of Tokyo, Tokyo, Japan.
Correspondence to Tsukasa Ohmori, MD, PhD, Research Division of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical University School of Medicine, 3111-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan. E-mail tohmori{at}jichi.ac.jp
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Abstract |
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Top
Abstract
IntroductionBackground and Purpose— We have previously shown that the sphingosine 1-phosphate (S1P)/S1P receptor-1 (S1P1R) axis contributes to the migration of transplanted neural progenitor cells (NPCs) toward areas of spinal cord injury. In the current study, we examined a strategy to increase endogenous NPC migration toward the injured central nervous system to modify S1PR.
Methods— S1P concentration in the ischemic brain was measured in a mouse thrombosis model of the middle cerebral artery. NPC migration in vitro was assessed by a Boyden chamber assay. Endogenous NPC migration toward the insult was evaluated after ventricular administration of the S1P2R antagonist JTE-013.
Results— The concentration of S1P in the brain was increased after ischemia and was maximal 14 days after the insult. The increase in S1P in the infarcted brain was primarily caused by accumulation of microglia at the insult. Mouse NPCs mainly expressed S1P1R and S1P2R as S1PRs, and S1P significantly induced the migration of NPCs in vitro through activation of S1P1R. However, an S1P1R agonist failed to have any synergistic effect on S1P-mediated NPC migration, whereas pharmacologic or genetic inhibition of S1P2R by JTE-013 or short hairpin RNA expression enhanced S1P-mediated NPC migration but did not affect proliferation and differentiation. Interestingly, administration of JTE-013 into a brain ventricle significantly enhanced endogenous NPC migration toward the area of ischemia.
Conclusions— Our findings suggest that S1P is a chemoattractant for NPCs released from an infarcted area and regulation of S1P2R function further enhances the migration of NPCs toward a brain infarction.
Key Words: migration • sphingosine 1-phosphate receptor-2 • sphingosine 1-phosphate • neural progenitor cells
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Introduction |
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Neural stem/progenitor cells (NPCs), self-renewing cells with the capacity to differentiate into neural cells, have been shown to exist mainly in 2 specific brain regions within the adult central nervous system (CNS): the subventricular zone (SVZ) and the hippocampal subgranular zone.1 NPCs proliferate in the SVZ and migrate through it in a pattern reminiscent of the rostral migratory stream toward the olfactory bulb, where they differentiate into mature neurons.2 NPCs also migrate to sites of brain injury. This may represent an adaptive response to limit or repair damage.3,4 Newly generated NPCs are recruited from the SVZ to nearby areas of neural damage, and some show region-specific differentiation, known as neurogenesis.3,4 A recent study showed that neurogenesis after brain injury is a meaningful response and that blockade of neurogenic cell division by irradiation worsens the outcome of cerebral ischemia.5 Thus, a precise understanding of the mechanism underlying injury-mediated NPC migration may contribute to improving the effectiveness of stem cell–based therapies for CNS disorders.
Recently, we reported the importance of sphingosine 1-phosphate (S1P), a lysophospholipid mediator, for injury-mediated NPC migration.6 S1P is currently attracting a great deal of attention as a bioactive sphingolipid that has various cellular functions and acts via the 7 transmembrane S1P receptors (S1PRs), S1P1R through S1P5R.7–9 We reported that the S1P concentration in the spinal cord increased after a contusion injury and contributed to the migration of transplanted NPCs in vivo via the S1P1R.6 In the current study, we show that S1P-mediated NPC migration toward an area of brain injury is enhanced by modulation of S1P2R function. We propose new therapeutic approaches to enhance the mobilization of endogenous NPCs toward areas of CNS injury.
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Materials and Methods |
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Materials, measurement of S1P, immunohistochemistry, methods for migration, cell proliferation, NPC differentiation, construction of a lentiviral vector, and reverse transcription–polymerase chain reaction (RT-PCR) are described in detail in the supplemental materials, available online at http://stroke.ahajournals.org.
Photochemically Induced Brain Infarction
All animal procedures were approved by the institutional animal care and concern committee at Jichi Medical University. Animal care was performed in accordance with the committee’s guidelines. C57BL/6 female mice (8 to 12 weeks old) were purchased from Japan SLC (Shizuoka, Japan). Thrombosis of the middle cerebral artery (MCA) in mice was performed as described.10 In brief, mice were anesthetized with 1.7% to 2.0% isoflurane and the temporal muscle was transected to expose the skull. The left distal MCA could be observed through the skull. Photoillumination was achieved with a xenon lamp (model L4887–03; Hamamatsu Photonics, Hamamatsu, Japan) via an optic fiber with a focus. The light was focused onto the MCA over the intact skull at a power of 2.3x106 lux. The photosensitizing dye Rose Bengal was simultaneously administered at a dose of 20 mg/kg IV within 5 minutes of laser irradiation.
Cell Culture
Mouse NPCs were isolated and cultured, as described previously.6 In brief, the forebrains of E14.5 mouse embryos were isolated and mechanically dissociated into a suspension of single cells. The dissociated cells were cultured in Dulbecco’s modified Eagle’s medium/F12 supplemented with B27 supplement (Invitrogen, Carlsbad, Calif), 20 ng/mL basic fibroblast growth factor, and 20 ng/mL endothelial growth factor. The cells were used for experiments between passages 2 and 4.
Infusion of JTE-013 Into Brain Ventricles
For studies involving pharmacologic blockade of S1P2R, 0.25 µL/h Alzet Minipumps (Durect Corp, Cupertino, Calif) were used for drug delivery. Empty pumps with flow moderators were weighed, filled with 1 mmol/L JTE-013 or phosphate-buffered saline (containing the same concentration of dimethyl sulfoxide as control), and then reweighed. Flow moderators were connected to a catheter (Alzet brain infusion kit 2; Durect Corp), which was connected to an infusion cannula. Pumps were stored overnight at 37°C in sterile saline to prime drug release. Two days after MCA thrombosis was induced, mice were anesthetized with isoflurane and placed in a small-animal stereotaxic frame. JTE-013 infusion during the acute phase of infarction may change the infarct size because inhibition of S1P2R is reported to affect vascular function.11,12 Hence, we started the JTE-013 infusion 2 days after ischemia. The scalp was shaved, cleaned, and opened with a scalpel. A small burr hole was drilled 1 mm lateral and 0.2 mm caudal from the bregma. The catheter was lowered into the left ventricle (depth, 2.5 mm ventral) and affixed with dental cement, and the opening was sutured shut. Mice were euthanized by decapitation after 14 days. Cannula placement was verified by direct observation by cutting an insertion point. Once confirming the correct insertion, we proceeded to histologic analysis. The transverse sections (0.5 mm forward of the bregma) were analyzed to assess NPC migration in vivo. Endogenous NPC migration was detected by immunostaining of NPCs with an anti-doublecortin (DCX) polyclonal antibody (Santa Cruz Biotechnology). The distance to the ischemic area from the SVZ was separated into 3 parts (0 to 300 µm, 301 to 600 µm, and 601 to 900 µm). The DCX-positive area in each part was quantified with the use of Scion Image for Windows (Scion Corp). For 3-dimensional counting of migrated cells, 3 separate sections (0.5 mm, 0.7 mm, and 0.9 mm forward of the bregma) were prepared for analyses. To count the number of DCX-positive cells, the number of nuclei stained with DAPI in the DCX-positive area was manually counted by a blinded observer and expressed as the total number of DCX-positive cells in each area.
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Results |
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Changes of S1P Concentration After Brain Infarction
To investigate the physiologic role of S1P in ischemic stroke, brain S1P concentrations were measured in a mouse model of brain ischemia. After brain infarction by photochemical induction of thrombosis in the distal MCA, most glial cells and neurons in the affected brain would be dead during the acute phase of infarction. Reflecting this cell deterioration, S1P content was significantly decreased 3 days after infarction (Figure 1A). Interestingly, S1P content in the ischemic brain was significantly increased thereafter and reached a maximum 14 days after ischemia (Figure 1A). On the other hand, changes in dihydro-S1P concentration after ischemia were marginal (Figure 1B). Immunohistologic analysis with an anti-S1P antibody confirmed that the site of infarction contained a large amount of S1P (Figure 1C). S1P was highly expressed at the boundary zone and in the central core of the infarct (Figure 1C).
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Next, we examined the cellular location of the elevated S1P content after brain infarction by immunohistochemistry. Destruction of the normal structure of the CNS and accumulation of microglia and immunoreactive cells of nonneural lineages expressing CD11b were observed after the insult (Figure 2A). S1P was highly expressed in the region of microglia accumulation in the infarct area and boundary zone (Figure 2A). On the other hand, astrocytes expressing glial fibrillary acid protein (GFAP) accumulated primarily at the boundary zone, whereas S1P immunoreactivity was partly colocalized, but S1P in the infarcted area was not (Figure 2B). As expected, few microtubule-associated protein 2 (MAP-2)–positive neurons were observed in the insult areas (data not shown). As well, most S1P immunoreactivity did not merge into B lymphocytes (B220) and T lymphocytes (CD3e; Figure 2C) in the infarcted area. These data suggest that microglia that accumulate at the site of injury are the main sources responsible for S1P elevation, whereas astrocytes might partly contribute to the increase in S1P in the boundary zone.
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Expression of S1PR in Mouse NPCs
Many if not all of the biologic responses induced by S1P are mediated by its cell surface receptors, ie, S1PRs (S1P1R through S1P5R).8,9 Next, we examined the expression of S1PRs in several adult mouse tissues and NPCs. Although S1P1R seemed to be ubiquitously expressed, the patterns of S1PR expression were quite different among the tissues examined (Figure 3A). In this study, the NPCs expressed all known S1PRs (Figure 3A); real-time, quantitative RT-PCR analysis of NPCs revealed that S1P1R and S1P2R were the most highly expressed (Figure 3B).
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Migration of NPCs Toward S1P
We next evaluated the effects of S1P on NPC migration. As shown in Figure 4A, S1P-induced migration resulted in a bell-shaped concentration-response curve, and the maximal response was observed at 100 nmol/L. To explore which S1PRs were involved in S1P-mediated NPC migration, we used 2 S1P-related synthetic compounds, VPC23019 and VPC24191. VPC23019 acts as a competitive inhibitor of S1P1R and S1P3R but partly stimulates S1P4R and S1P5R.13 On the other hand, VPC24191 is an agonist against S1P1R and S1P3R. VPC23019 failed to induce NPC migration and abolished migration toward S1P (Figure 4B). VPC24191 by itself dramatically enhanced NPC migration in a concentration-dependent manner (Figure 4C). Interestingly, the addition of S1P inhibited the NPC migration elicited by a higher concentration of VPC24191 (1 to 10 µmol/L; Figure 4C). These data suggest that S1P1R is the primary receptor involved in S1P-mediated NPC migration, in support of our previous study,6 but also indicated that other S1PRs can regulate the S1P1R-mediated response.
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We next focused on S1P2R, a receptor that is responsible for inhibition of migratory responses.14,15 Although JTE-013, a specific antagonist of S1P2R, had no effect on NPC migration, S1P-mediated migration modulated by JTE-013 reached that induced by VPC24191 (Figure 4D). Similar results were observed after expression of short hairpin (sh) RNA: short interfering RNA against S1P1R (S1P1B) inhibited S1P-mediated NPC migration, whereas S1P2R knockdown (S1P2A) significantly enhanced the migration of NPCs elicited by S1P (Figure 4E). Considering that the concentration of S1P increased in the ischemic area, modulation of S1P2R instead of S1P1R, could be a more practical approach to increase the mobilization of NPCs.
To examine whether inhibition of S1P2R affects other stem cell properties, we examined the effects of JTE-013 on the proliferation and differentiation of NPCs. S1P reportedly induced cell differentiation and proliferation16; however, S1P had only a marginal effect on NPC proliferation and differentiation in this study (Figure 5), and JTE-013 was without effect (Figure 5). These data suggest that modulation of S1P2R enhances S1P-mediated migration of NPCs without the loss of cell viability and the potential to differentiate.
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Involvement of S1P2R in Endogenous NPC Migration
Finally, we attempted to increase the mobilization of endogenous NPCs by S1P2R antagonism. After infarction, we analyzed NPC migration from the lateral ventricle wall by immunofluorescence against DCX, a microtubule-associated protein that is specifically expressed in NPCs and immature neurons. We started continuous administration of JTE-013 into the ventricle 2 days after focal brain ischemia to examine whether targeting S1P2R would enhance endogenous NPC migration toward the area of the insult (Figure 6A). NPCs expressing DCX migrated from the lateral ventricle wall and accumulated around the ischemic area (Figure 6B). Interestingly, NPC migration toward the ischemic area was dramatically enhanced by ventricular infusion of JTE-013 (Figures 6B through 6D). Administration of JTE-013 into ventricles could diffuse into many areas in the brain and so might affect many neurologic functions; many kind of neural cells reportedly express S1PRs, including S1P2R.17 However, enhanced NPC migration after administration of JTE-013 was not observed in the contralateral side of the infarct (data not shown). These data suggest that pharmacologic inhibition of S1P2R in NPCs by JTE-013 promotes endogenous NPC migration toward the areas of brain ischemia where S1P has increased.
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Discussion |
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Migration of NPCs is important not only for development of the embryonic nervous system but also for repair of the nervous system after injury.3,4 Identifying candidate molecules that could play a role in NPC migration is crucial for understanding proper tissue formation by newly formed neural cells, as well as for developing novel therapies to promote neural repair after CNS injury.18,19 We previously showed a role for S1P in NPC migration toward a pathologic area of the CNS and proposed that elevation of S1P at the site of injury was a guiding factor for NPC migration.6 Here, we reveal that S1P2R is a potential target for strategies aiming to increase NPC migration after brain ischemia.
We have already shown that S1P1R contributes to NPC migration toward areas of high S1P expression in the injured CNS.6 This occurs in a variety of cell types through Gi-mediated Rac activation,20,21 whereas S1P2R abolishes migration after the coupling of S1P2R to the G12/13/Rho pathway.11,22 The S1P2R abolished migration not only through S1P1R but also other types of receptors for growth factor.14,15 Overactivation of the Rho/Rho-kinase pathway is thought to be 1 of the important mechanisms that strongly inhibit cell migration.11 These data present 2 strategies for the induction of NPC migration to a site of injury: elevation of S1P1R signaling or blockade of S1P2R signaling. However, the S1P1R-specific agonist failed to enhance NPC migration in the presence of S1P, suggesting that activation of S1P1R itself could not overcome the inhibitory effect of S1P2R in NPCs. Considering that the concentration of S1P increases at the site of an insult, modulation of S1P2R function could be a more practical approach for the mobilization of NPCs when compared with modulation of S1P1R function. We confirmed the involvement of S1P2R in in vitro NPC migration by RNA interference experiments; however, the effects of JTE-013 unrelated to S1P2R antagonism could not be completely eliminated because JTE-013 is reported to inhibit vasoconstriction through unknown mechanism(s) independent of the S1P2R.23 Further studies with gene-deficient mice would be required to confirm the full effects of S1P2R inhibition.
Migration of NPCs is an important process in neurogenesis.4 The neural stem cells of the SVZ are the principal source of NPCs, which form chainlike structures and migrate laterally toward the injured striatal regions before differentiating into mature neurons.3,4,19 Our data suggest that local elevation of S1P concentration after brain ischemia acts as an activation signal for NPCs in the SVZ and that modulation of S1P2R function could enhance endogenous NPC migration. Recent studies have suggested that endogenous NPC migration and angiogenesis are mechanistically linked in the nervous system; neuroblast cells migrate toward blood vessels in areas undergoing early vascular remodeling.24,25 Vascular endothelial growth factor, a major angiogenic factor that acts as a guiding factor for endothelial progenitors, is an attractive guidance cue for the migration of undifferentiated NPCs.26,27 Our data show that S1P, another important angiogenic factor,20 also guides the migration of NPCs, suggesting a mechanistic link between NPC migration and angiogenesis via the S1P/S1PR axis in the nervous system.
The change in S1P concentration after brain ischemia is quite different from that of known inflammatory cytokines, chemokines, and growth factors. Intercellular adhesion molecules, including intercellular adhesion molecule-1 and selectins, are rapidly induced 3 to 6 hours after ischemia and peak at 6 to 12 hours.28,29 The expression of adhesion molecules on microvessels promotes neutrophil recruitment and trafficking into the brain.28,29 Adhesion molecules and inflammatory mediators play a role in focal ischemic brain injury. In this study, the concentration of S1P was gradually enhanced at the site of ischemia, and S1P was highly expressed at the site of microglia accumulation. The gradual increase in S1P led us to postulate that it plays an important role in regeneration after CNS injury. Microglia reportedly release an unidentified chemoattractant(s) for NPCs after CNS injury and thus play an important role in directing the replacement of damaged or lost cells in the CNS.30 Recently, selective ablation of microglial cells was shown to exacerbate ischemic injury in the brain, suggesting that microglial cells serve as an endogenous pool of neurotrophic molecules.31 Because S1P enhances the migration of NPCs, we postulate that S1P is a physiologic, neuroprotective substance released from microglia.
An issue that remains to be addressed is what stimulates S1P biosynthesis after injury. A common product of sphingolipid breakdown is ceramide, which is generated primarily by hydrolysis of membrane sphingomyelin.32 Ceramide can be further catabolized by ceramidases to generate sphingosine, which can be phosphorylated through the action of sphingosine kinase, generating S1P.33 Sphingosine kinase activity in the brain was recently reported to increase 24 hours after ischemia in a mouse model of MCA occlusion34; however, those increases were not necessarily the same as the S1P concentration in our study. As well, if elevation of sphingosine kinase activity accounts for the increase in S1P concentration after insult, the concentration of dihydro-S1P would increase because dihydrosphingosine is phosphorylated by sphingosine kinase isozymes to an extent similar to that of naturally occurring sphingosine.35,36 One possible mechanism is sphingomyelin breakdown after phagocytosis of neural cells by activated microglia. Microglial activation and phagocytic potential are reported to occur gradually between 1 and 12 weeks after an ischemic insult.37 Activated microglial cells phagocytose dead neural cells and may induce sphingomyelin breakdown during the neuroregeneration phase. Investigations into the precise mechanisms of injury-mediated S1P elevation and sphingosine metabolism at injury sites are now under way in our laboratory.
Summary
In summary, increased S1P at the site of brain infarction acts as a chemoattractant for NPCs. Although activation of S1P1R failed to enhance NPC migration in the presence of S1P, S1P2R, a specific antagonist of the migration-inhibitory receptor, upregulates migration responses induced by S1P and augments endogenous NPC migration toward the ischemic insult. These data suggest that S1P2R blockade is a promising candidate to enhance NPC migration toward sites of brain infarction. Further studies in gene-deficient mice will be needed, as will behavioral and functional analyses after the administration of S1P2R antagonists for treatment of ischemic stroke.
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Acknowledgments |
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We thank N. Matsumoto, M. Ito, and A. Ishiwata for their excellent technical assistance.
Sources of Funding
This work was supported by the Takeda Science Foundation; grants-in-aid for scientific research from the Ministry of Education and Science; Health and Labour science research grants for research from the Ministry of Health, Labour and Welfare; and grants for High-Tech Center Research projects for private universities, with a matching fund subsidy from the Ministry of Education, Culture, Sports, Science, and Technology, 2002–2006.
Disclosures
None.
Received January 14, 2008; revision received April 21, 2008; accepted April 22, 2008.
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Supplemental Materials and Methods
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Materials |
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The following materials were obtained from the indicated suppliers: anti-S1P mouse monoclonal antibody (clone NHS1P; Alfresa Pharma Corp, Tokyo, Japan); anti-DCX polyclonal antibody (clone H-280; Santa Cruz Biotechnology Inc, Santa Cruz, Calif); synthetic S1P-related compounds VPC23019 and VPC24191 (Avanti Polar Lipids Inc, Alabaster, Ala)1; biotin-conjugated anti-CD3e (clone 145-2C11) and anti-B220 (clone RA3-B62; BD Biosciences, San Jose, Calif); and the specific S1P2R antagonist JTE-013 (Merck Chemicals Ltd, Darmstadt, Germany).2,3 Other materials, cytokines, and antibodies used in this study were from previously described sources.4
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Measurement of S1P |
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After excision of a 5-mm transverse cross section of the ischemic brain after an indicated time after infarction, the sections were separated into ipsilateral (infarction) or contralateral (control) sides. We separately measured S1P concentration in each side. Three milliliters of ice-cold chloroform/methanol (1:2) was added to each section and the samples were sonicated. Lipids were extracted from the samples, and the S1P concentrations measured by reaction with o-phthalaldehyde as described previously.4 To simultaneously measure S1P and dihydro-S1P, C17-sphingosine 1-phosphate was used as an internal standard.5
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Immunohistochemistry |
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Samples were fixed with 4% p-formaldehyde in phosphate-buffered saline (PBS) for 2 hours at 4°C, incubated with PBS containing sucrose (10% to 30%), and then frozen in the presence of OCT compound in dry ice/ethanol. Sections were prepared from frozen tissues at –25°C and placed on polylysine-coated glass slides. For detection of S1P localization by immunohistochemistry, tissue sections were blocked with MOM mouse immunoglobulin blocking reagent (Vector Laboratories, Burlingame, Calif) and 5% donkey serum in PBS containing 0.2% Triton-X 100. Primary antibody detection was performed by biotinylated antimouse IgM antibody (Vector Laboratories) and horseradish peroxidase–conjugated streptavidin and visualized with Vectastain ABC kit (Vector Laboratories). Immunofluorescent staining was performed as described previously4 and was observed with a confocal microscope (FV1000, Olympus, Tokyo, Japan).
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Migration |
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NPC migration was assessed with a modified Boyden chamber assay, as described previously.4 Polycarbonate filters with 8-µm pores, used to separate the upper and lower chambers, were coated with 10 µg/mL collagen type IV (Becton-Dickinson). NPCs were added to the upper chamber at a density of 1x105/100 µL medium containing 1% fatty acid–free bovine serum albumin and incubated for 12 hours at 37°C. The NPCs were allowed to migrate toward the indicated chemoattractant in the lower chamber. After the reaction, the filters were fixed and subjected to Giemsa staining. After removal of nonmigrated cells by wiping with cotton swabs, cells that had migrated through the filter to the lower surface were counted manually under a microscope in 5 predetermined fields at a magnification of 100x.
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Cell Proliferation |
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NPC proliferation was assessed by bromodeoxyuridine (BrdU) incorporation into cells (BrdU flow kit, BD Biosciences). When BrdU was directly added to the neurosphere culture after growth factor depletion, a number of dead cells were observed. To preserve cell viability after growth factor depletion, we changed the culture condition of NPCs as adherent cells. In brief, NPCs (3x105 cells) were seeded onto 12-well culture plates coated with 1 µg/mL fibronectin (Sigma Aldrich) in the presence of B27 supplement, 20 ng/mL endothelial growth factor, and 20 ng/mL basic fibroblast growth factor. After 12 hours, the cells were starved of growth factors for 3 hours before 10 nmol BrdU was added to the medium. Cell attachment onto fibronectin allowed cell viability to be preserved even with the depletion of growth factor (data not shown). However, the longer depletion of growth factor resulted in an increase in apoptotic cells, which reduced the basal incorporation of BrdU in NPCs. The proportion of apoptotic cells in our procedure ranged from 1.9% to 6.0%. Cells were stimulated with the indicated agonist for 1 hour, and BrdU incorporation then analyzed by flow cytometry in accordance with the manufacturer’s instructions (BrdU flow kit, BD Biosciences).
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NPC Differentiation |
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NPC differentiation was induced in the absence of growth factors and the presence of 1% bovine serum for 5 days. NPCs (5x104 cells) were seeded onto 10 µg/mL poly-L-ornithine–coated 8-well chamber slides (Nalge Nunc International, Rochester, NY). Growth factors were depleted, and cells were stimulated with 1% fetal bovine serum and an indicated agent(s) to promote differentiation. The medium was exchanged every other day. Differentiation was assessed by immunofluorescent staining. Cells were fixed with PBS containing 4% p-formaldehyde and then blocked with 5% donkey serum and 0.3% Triton X-100 for 2 hours. After being washed with PBS, neurons and astrocytes were simultaneously stained by neuron-specific anti-βIII tubulin (Tuj-1) monoclonal antibody (1:500; Sigma-Aldrich Corp, St. Louis, Mo) and astrocyte marker anti-glial fibrillary acidic protein rabbit polyclonal antibody (1:500; Chemicon International Inc, Temecula, Calif). Antibody binding was detected with Alexa 488–conjugated anti-mouse IgG antibody (1:200, Invitrogen) and Alexa 594–conjugated anti-rabbit IgG (1:200, Invitrogen). Oligodendrocytes were detected by an anti-oligodendrocyte marker O4 antibody (1:500; R&D Systems Inc, Minneapolis, Minn). Antibody binding was detected with Alexa 488–labeled anti-mouse IgM antibody (1:200, Invitrogen). Samples were mounted in Vectorshield with DAPI (Vector Laboratories) and observed by confocal microscopy (FV1000, Olympus, Tokyo, Japan).
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Construction of a Lentiviral Vector and Virus Transduction |
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A gene transfer vector, pLL3.7, for constructing a replication-defective self-inactivating HIV short hairpin RNA vector was purchased from American Type Culture Collection. Putative short interfering RNA (siRNA) sequences were designed with web-based software provided by Dharmacon RNA Technologies. We designed 2 siRNA sequences for each target and selected the sequence with the greatest knockdown of mouse S1PR expression (data not shown). The siRNA sequences for S1P1R (S1P1-B sequence) and S1P2R (S1P2-A sequence) corresponded to nucleotides 406 to 424 of mS1P1R (GeneBank No. BC_051023) and 321 to 339 of mS1P2R (No. BC_096760), respectively. Lentiviral vector and viral transduction was performed according to our reported methods.4
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Reverse Transcription–Polymerase Chain Reaction |
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RT-PCR and real-time RT-PCR were performed as described.4 The oligonucleotide primer pairs used in this study were as follows: 5'-ACTATATTCTCTTCTGCACCAC-3' (sense) and 5'-GCTTCGAGTCCTGACCCA-3' (antisense) for S1P1 (No. BC_051023); 5'-TGTCACTCTGTCCTTAACTC-3' (sense) and 5'-GGCCACTTGTCTCTCGAT-3' (antisense) for S1P2 (No. BC_096760); 5'-CAACTTGGCTCTCTGCGACCT-3' (sense) and 5'-ACTGTTGGAGACAGACTGAACG-3' (antisense) for S1P3 (No. BC_068176); 5'-CTCTACTCCAAGGGCTATGT-3' (sense) and 5'-TGGAGACTTCTGCCCATT-3' (antisense) for S1P4 (No. BC_107000); 5'-GTGTGTGCCTTCATTGTG-3' (sense) and 5'-CAGGTCCGACAAAGTGAG-3' (antisense) for S1P5 (No. BC_012232).
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References |
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4. Kimura A, Ohmori T, Ohkawa R, Madoiwa S, Mimuro J, Murakami T, Kobayashi E, Hoshino Y, Yatomi Y, Sakata Y. Essential roles of sphingosine 1-phosphate/S1P1 receptor axis in the migration of neural stem cells toward a site of spinal cord injury. Stem Cells. 2007; 25: 115–124.[CrossRef][Medline] [Order article via Infotrieve]
5. Min JK, Yoo HS, Lee EY, Lee WJ, Lee YM. Simultaneous quantitative analysis of sphingoid base 1-phosphates in biological samples by o-phthalaldehyde precolumn derivatization after dephosphorylation with alkaline phosphatase. Anal Biochem. 2002; 303: 167–175.[CrossRef][Medline] [Order article via Infotrieve]
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