J. Clin. Invest. = 113:=20 1364-1374 (2004). doi:10.1172/JCI20001.
Copyright = =C2=A92004 by the=20 American Society for Clinical Investigation

Research Article

Neuronally expressed stem cell = factor=20 induces neural stem cell migration to areas of brain = injury

Lixin Sun, Jeongwu = Lee=20 and Howard A. Fine

Neuro-Oncology Branch, National = Cancer=20 Institute, National Institutes of Neurological Disorder and = Stroke,=20 NIH, Bethesda, Maryland, USA.

Published May 1, 2004
Received = for=20 publication September 9, 2003, and accepted in revised form = February=20 17, 2004.

Abstract

Neural stem/progenitor cell (NSPC) migration toward sites = of=20 damaged central nervous system (CNS) tissue may represent an = adaptive response for the purpose of limiting and/or = repairing=20 damage. Little is known of the mechanisms responsible for = this=20 migratory response. We constructed a cDNA library of injured = mouse=20 forebrain using subtractive suppression hybridization (SSH) = to=20 identify genes that were selectively upregulated in the = injured=20 hemisphere. We demonstrate that stem cell factor (SCF) mRNA = and=20 protein are highly induced in neurons within the zone of = injured=20 brain. Additionally, the SCF receptor c-kit is expressed on = NSPCs in=20 vitro and in vivo. Finally, we demonstrate that recombinant = SCF=20 induces potent NSPC migration in vitro and in vivo through = the=20 activation of c-kit on NSPCs. These data suggest that the = SCF/c-kit=20 pathway is involved in the migration of NSPCs to sites of = brain=20 injury and that SCF may prove useful for inducing progenitor = cell=20 recruitment to specific areas of the CNS for cell-based = therapeutic=20 strategies.

Introduction

Neural stem/progenitor cell (NSPC) migration is an = essential=20 process for the development of the central nervous system = (CNS) as=20 well as the ongoing neurogenesis that occurs in the mature = CNS of=20 most vertebrate species, including mammals (1=E2=80=933). It = has been=20 demonstrated that NSPCs proliferate in the subventricular = zone (SVZ)=20 and migrate tangentially through the SVZ, in a pattern = reminiscent=20 of the rostral migratory stream (RMS), toward the olfactory = bulb=20 where they differentiate into mature neurons (4=E2=80=936). It = has also=20 been recently demonstrated that NSPCs migrate to sites of=20 pathological insult such as various types of brain injury = (i.e.,=20 ischemia and blunt trauma) and tumors (7=E2=80=9312). = NSPC=20 migration toward damaged CNS tissue may represent an = adaptive=20 response for the purpose of limiting and/or repairing = damage,=20 although to date there are few data to definitively support = or=20 refute this hypothesis. Regardless of their physiological = role in=20 injury, the migratory properties of NSPCs could = theoretically be=20 exploited for cell-based therapeutics (13, = 14). = Thus, the=20 microenvironmental conditions and guidance signals that = regulate=20 NSPC migration in the adult need to be elucidated.

Although little is understood about the mechanism = controlling=20 NSPC migration in the adult CNS, several key mechanisms = responsible=20 for NSPC migration in embryonic development have been = elucidated.=20 Embryonic NSPCs recognize cues provided by cells along the = path of=20 migration and are guided at long distances by gradients of=20 chemoattractant molecules that are released selectively by = cells=20 along the way and at their final destination (15=E2=80=9317). = The=20 discovery of netrins (18=E2=80=9321), = semaphorins=20 (22=E2=80=9324), = ephrins=20 (25, = 26), = and Slit=20 proteins (27=E2=80=9329) = established=20 the existence of neuronal migration guidance signals at long = distances by diffusible secreted proteins that have NSPC=20 chemoattractant and repulsive properties. Recently it has = been=20 suggested that certain cytokines with known important = functions in=20 CNS development (i.e., transforming growth factor =CE=B1, = bFGF, and EGF)=20 were capable of enhancing ischemia-induced progenitor = proliferation=20 and migration (30, = 31).=20 Nevertheless, our understanding of the mechanisms guiding = neural=20 progenitor cell migration in pathological states remains = limited.=20 Identification of diffusible NSPC chemoattractant factors = would not=20 only help to better understand the totality of mechanisms=20 responsible for injury-mediated NSPC migration but also = could have=20 significant therapeutic implications for the prospect of = cell-based=20 therapeutic strategies for injury repair.

In an attempt to identify NSPC chemoattractant molecules = in=20 injured brain tissue, we utilized subtractive cDNA = suppression=20 hybridization (SSH) to identify genes that were selectively=20 upregulated during injury. We then utilized an in vitro NSPC = migration assay to screen the products of our isolated cDNA = clones=20 for NSPC chemotactic activity. We now report the = identification of=20 stem cell factor (SCF) as a cytokine that is highly = overexpressed by=20 neurons at sites of brain injury. We additionally show that = the SCF=20 receptor (c-kit) is expressed on NSPCs and is = autophosphorylated=20 following ligand binding and that SCF mediates potent=20 chemoattractant activity for NSPCs both in vitro and in = vivo.

Methods

Animals and =E2=80=9Cfreeze=E2=80=9D injury. = All mice=20 (NCI-Frederick, Frederick, Maryland, USA) were handled = following=20 Institutional guidelines (National Institutes of Health = ethics=20 guidelines for use of animals in research). Stereotactic = surgery was=20 performed with SCID male mice (6=E2=80=938 weeks of age) = sedated by=20 anesthesia (ketamine and xylazine at doses of 80 mg/kg body = weight=20 and 10 mg/kg body weight, respectively, by intraperitoneal=20 administration). As schematically illustrated in the Figure = 1A, the skull = was opened=20 1 mm anterior to the bregma and 2.5 mm lateral to the = midline with a=20 dental drill, leaving the dura intact. The = =E2=80=9Cfreeze=E2=80=9D brain injury was=20 produced by insertion of a Hamilton syringe precooled in = liquid=20 nitrogen through a cranial hole to a depth of 2 mm below the = dural=20 surface. The needle was kept in this position for 30 = seconds. This=20 procedure was repeated five times in each animal. The = animal=E2=80=99s body=20 temperature was kept within physiological range during and = after the=20 surgery. The animals were euthanized at 5 days after injury = by=20 CO₂ inhalation and their brains were rapidly = dissected on=20 an ice-cold board. The dorsal forebrain ipsilateral and=20 contralateral to the site of injury was rapidly dissected = out=20 (illustrated in Figure 1A) and stored = at =E2=80=9370=C2=B0C=20 for RNA extraction. The entire dorsal forebrain from animals = with=20 bilateral injury was collected for Western blot and the = Boyden=20 chamber migration assay. In these studies, the corresponding = brain=20 areas from naive animals were used as controls.

Figure=20 1

SCF induced by =E2=80=9Cfreeze=E2=80=9D injury to the = brain. (A) Schematic=20 illustration of =E2=80=9Cfreeze=E2=80=9D injury. Blue arrow = indicates the insertion=20 track of the precooled needle. Red lines separate the = forebrain into=20 injured and uninjured hemispheres and divide the hemispheres = into=20 its dorsal and ventral halves. CX, cortex; CPu, caudate = putamen=20 (striatum); CC, corpus callosum; LV, lateral ventricle. = (B)=20 Custom microarray confirms increased SCF message in = the=20 injured brain at day 5. The cDNA clones from SSH library = were loaded=20 individually onto duplicate nitrocellulose filters, one = hybridized=20 by the injured brain_derived cDNAs (Injury) and another = hybridized=20 by contralateral control (Control). Membranes with the = SCF=20 gene are shown, and the SCF spots are marked with red = and=20 blue circles. Actin and GAPDH are shown as = green and=20 black circles, respectively. SCF images are stronger in the = injured=20 brain_derived cDNA_hybridized blot than the control. = (C)=20 Quantitative RT-PCR. Fold induction of SCF mRNA in=20 =E2=80=9Cfreeze=E2=80=9D-injured forebrain 5 days after = injury compared with the=20 uninjured contralateral side (P < 0.05). The = SCF=20 mRNAs were individually normalized to levels of 18S RNA. = (D)=20 Time-dependent changes in SCF protein induction after = =E2=80=9Cfreeze=E2=80=9D brain=20 injury, as measured by Western blot, using uninjured = forebrain as a=20 control. Two major bands were present, representing the=20 transmembrane form of SCF (33 kDa) and the cleaved, soluble = form of=20 SCF (19 kDa). (E) Quantification of SCF expression by = computer densitometry. The band intensities of SCF were = normalized=20 to those of =CE=B2-tubulin.

Cell culture. Human NSPCs (Clonetics, = Walkersville,=20 Maryland, USA) and mouse NSPCs isolated from the forebrains = of=20 embryos at day 14.5 of gestation were cultured in neural = basal=20 medium supplemented with B27 (Invitrogen Corp., Carlsbad,=20 California, USA), 20 ng/ml of bFGF and 20 ng/ml of EGF = (R&D=20 Systems, Minneapolis, Minnesota, USA), 0.5 =E2=88=9DM = glutamine, and=20 appropriate antibiotics, as described previously (32). = The cells=20 grew in a six-well plate as nonadherent cells and were = prevented=20 from attaching to the plates by periodic gentle agitation of = the=20 plates each day. EGF and bFGF were added every other day and = culture=20 medium was changed weekly until neurospheres became visible. = The=20 spheres were passaged by enzymatic and mechanical = dissociation every=20 7=E2=80=9310 days and were reseeded as single cells into = growth medium at a=20 density of about 100,000 cells/ml. The cells that adhered to = the=20 plastic and began to extend processes were removed from the = wells=20 and were not cultured in subsequent passages. U-87, a cell = line=20 derived from a malignant glioma, and NIH 3T3 were purchased = from=20 American Type Culture Collection (Manassas, Virginia, USA) = and were=20 cultured in the conditions recommended by the company.

Boyden chamber assay. The effects of SCF on = NSPC=20 migration were determined by a modified Boyden chamber assay = as=20 described previously (33). A = 96-well=20 cell migration kit was utilized (Chemicon, Temecula, = California,=20 USA), and each well was separated into two chambers by a = membrane=20 with 8-=E2=88=9Dm pores. The cells were briefly trypsinized = and were=20 adjusted to a density of 4 =E2=88=9E 10⁵ cells/ml = in a 1:20=20 dilution of B27 medium without growth factors. Some cells = were=20 pretreated with a c-kit=E2=80=93blocking antibody (ACK45; = PharMingen, San=20 Diego, California, USA) for 30 minutes at room temperature.=20 Migration of NIH 3T3 cell was also assayed as a control. = Recombinant=20 mouse SCF (rmSCF) (R&D Systems) was dissolved in the = same medium=20 and adjusted to the various concentrations. After different=20 concentrations of SCF or brain tissue lysates were added to = the=20 lower chamber as attractants, 100 =E2=88=9Dl of the NSPCs (4 = =E2=88=9E=20 10⁴) was applied to the upper chamber on the top = of=20 membrane. A group of control chambers without rmSCF was also = included. After 4 hours of incubation at 37=C2=B0C, the = migratory cells=20 on the bottom of the insert membrane were dissociated from = the=20 membrane by incubation with cell detachment buffer. These = cells were=20 subsequently lysed and stained with CyQuant GR dye = (Chemicon), which=20 exhibits strong fluorescence enhancement when bound to = cellular=20 nucleic acids, and fluorescence was measured with a = fluorescence=20 plate reader with a 480/520-nm filter set.

SSH library. Total RNA was isolated from = either the=20 injured or contralateral dorsal forebrain from 60 mice using = Trizol=20 (Invitrogen Corp.) and was purified with an RNeasy kit = (Qiagen,=20 Valencia, California, USA). The poly(A) RNA was subsequently = isolated from purified total RNA with a poly(A) RNA = isolation kit=20 (Qiagen). Complementary DNA (cDNA) was synthesized from 2 = =E2=88=9Dg poly(A)=20 RNA using the SMART PCR cDNA Synthesis Kit and subtractive=20 hybridization was performed with the CLONTECH PCR-Select = cDNA=20 Subtraction Kit (BD Biosciences, Palo Alto, California, USA) = based=20 on the protocols suggested by the company. The = =E2=80=9Ctester=E2=80=9D cDNA came=20 from injured brain, whereas the =E2=80=9Cdriver=E2=80=9D = pool was from uninjured=20 forebrain. After adaptor ligation, the = =E2=80=9Ctester=E2=80=9D cDNA pool was then=20 hybridized with =E2=80=9Cdriver=E2=80=9D cDNAs at a ratio of = 1:20 for selection of=20 transcripts specifically upregulated in the injured = hemisphere.=20 After hybridization, suppression PCR using primers specific = for the=20 =E2=80=9Ctester=E2=80=9D PCR adaptors selectively amplified = differentially expressed=20 transcripts for 15 cycles. The amplified cDNAs were = subsequently=20 cloned into the T/A cloning vector pGEM-T Easy (Promega, = Madison,=20 Wisconsin, USA), and about 500 colonies were obtained in = total.

Custom cDNA microarray. To confirm that the = cDNA=20 clones of the SSH library contained the upregulated = transcripts=20 induced by the injury, custom cDNA microarrays were = constructed and=20 used to screen the library. First, individual colonies from = the SSH=20 library were grown overnight at 37=C2=B0C in Luria broth = medium=20 containing 50 =E2=88=9Dg/ml of ampicillin. After the = plasmids were isolated,=20 the inserts were cut out with the EcoRI restriction = enzyme=20 and were analyzed by electrophoresis on a 1% agarose gel. = Inserts=20 within the clones (=E2=88=BC85% of total) were then = amplified by PCR. The=20 PCR products were then =E2=80=9Cdot-blotted=E2=80=9D onto = GeneScreen Plus=20 hybridization transfer membranes (Perkin Elmer Life Sciences = Inc.,=20 Boston, Massachusetts, USA) in duplicate with the S&S = Minifold I=20 Dot-Blot System (Schleicher & Schuell, Dassel, Germany). = In=20 addition to the SSH library clones, each blot also contained = actin=20 and GAPDH as controls. The cDNA targets were then labeled = and=20 hybridized to the filter. The poly(A) RNA from brain 5 days = after=20 injury and uninjured brain was = =E2=80=9Creverse-transcribed=E2=80=9D into cDNA and=20 was amplified for 10 cycles with the SMART PCR cDNA = Synthesis Kit as=20 described above. Purified cDNA probes (100 ng per blot) were = labeled=20 with fluorescence by the random primer method using the Gene = Images=20 Random Prime Labelling and Detection System (Amersham = Pharmacia=20 Biotech, Piscataway, New Jersey, USA). Each duplicated = membrane was=20 hybridized with the injured or uninjured = brain=E2=80=93derived probes at=20 60=C2=B0C overnight. The subsequent washing and = antibody-mediated=20 detection was identical to the protocol recommended by the = company.=20 Finally, the hybridized membranes were laid next to each = other,=20 exposed to Kodak x-ray film (Eastman Kodak Co., Rochester, = New York,=20 USA), and developed simultaneously.

DNA sequencing and sequence analysis. Our = custom=20 cDNA microarray screen confirmed the presence of over 300=20 overexpressed clones in the SSH library. These clones were = then=20 sequenced using the ABI PRISM Dye Terminator Cycle = Sequencing Ready=20 Reaction kit (Perkin Elmer Life Sciences) with the T7 = promoter=20 primer on an ABI 373 automated sequencer (Applied = Biosystems, Foster=20 City, California, USA). Database homology searches for the = cDNA=20 sequence and deduced protein sequences were performed by a = basic=20 local alignment search tool program using the DNA and = expressed=20 sequence tag DNA databases at the National Center for = Biotechnology=20 Information (http://www.ncbi.nih.gov/BLAST). From the 300 = clones,=20 196 unique genes were identified as being upregulated in the = injured=20 area of the forebrain.

RT-PCR and real-time PCR. The reverse = transcription=20 reaction and PCR were performed as described previously = (34). = The=20 c-kit primers for mouse were = 5=E2=80=B2-CCATGTGGCTAAAGATGAAC-3=E2=80=B2=20 (upstream) and 5=E2=80=B2-ACTGCTGGTGCTCGGGTTTG-3=E2=80=B2 = (downstream), and for=20 human were 5=E2=80=B2-TATACAACCCTGGCATTATGTCC-3=E2=80=B2 = (upstream) and=20 5=E2=80=B2-TGCGAAGGAGGCTAAACCTA-3=E2=80=B2 (downstream). For = real-time RT-PCR=20 analysis, SCF probes were selected in the connecting = region=20 of two exons (5=E2=80=B2-ACTCTAGCGTGTAAATC-3=E2=80=B2); the = upstream primer was=20 5=E2=80=B2-GAAGTCAGTCTTTTCCCTTGACAGT-3=E2=80=B2 and the = downstream primer was=20 5=E2=80=B2-GCATGTCACATTATACTATTGCAAACA-3=E2=80=B2. Real-time = PCR was performed with=20 the ABI PRISM 7900 HT Sequence Detection System (Applied = Biosystems)=20 in triplicate and data were analyzed on the basis of = threshold cycle=20 values of each sample and were normalized with 18S RNA.

Immunoprecipitation and immunoblot. Injured = dorsal=20 forebrains were dissected 24 hours, 3 days, 5 days, 7 days = and 12=20 days after injury and were homogenized in a lysis buffer = containing=20 50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 0.5% NP-40, and = Protease=20 Inhibitor Cocktail (Roche, Indianapolis, Indiana, USA). = Brain tissue=20 was kept on ice for 30 minutes and supernatants were = collected after=20 centrifugation. The brain lysates were separated by = 4=E2=80=9312%=20 polyacrylamide gel electrophoresis (Invitrogen Corp.) and = were=20 transferred to membranes. The filters were blotted with = rabbit=20 polyclonal anti-SCF (1:50 dilution; Chemicon). The NSPCs = were=20 treated with 100 ng/ml of rmSCF or were left untreated for = 10=20 minutes and then were washed with ice-cold PBS. Some mouse = NSPCs=20 were treated with rat monoclonal c-kit=E2=80=93blocking = antibody (ACK45;=20 PharMingen) for 30 minutes at room temperature before SCF=20 stimulation. The NSPCs were resuspended in a protease = inhibitor=20 mixture containing radioimmunoprecipitation assay buffer (1% = NP-40,=20 0.5% sodium deoxycholate, 0.1% SDS, 0.1 mg/ml PMSF, and 0.1 = mM=20 NaVO₄ in PBS). The cell lysates were precleared = with=20 protein A=E2=80=93Sepharose (Amersham Pharmacia Biotech), = incubated with=20 rabbit polyclonal antibody against c-kit (clone H-300; Santa = Cruz=20 Biotechnology, Santa Cruz, California, USA), and collected = on=20 protein A=E2=80=93Sepharose beads. After being washed and = eluted, the=20 immunoprecipitates were separated by electrophoresis through = 7%=20 polyacrylamide gels. After transfer, filters were hybridized = with=20 anti-phosphotyrosine (1:1,000 dilution; clone PY99; Santa = Cruz=20 Biotechnology) and visualized with a peroxidase-conjugated = secondary=20 antibody. After detection of SCF or a phosphotyrosine within = c-kit,=20 the membranes were stripped and reblotted with = =CE=B2-tubulin antibody=20 (Santa Cruz Biotechnology) or with anti=E2=80=93c-kit = (H-300; Santa Cruz=20 Biotechnology).

Immunocytochemistry. Cells and brain = sections were=20 processed for immunocytochemistry as described previously = (35). = Briefly,=20 brains were fixed in 4% phosphate-buffered paraformaldehyde = (PFA)=20 after transcardial perfusion and were processed as paraffin = sections=20 or floating sections. For paraffin sections, brains were = embedded in=20 paraplast and cut into coronal sections 10 =E2=88=9Dm in = thickness on a=20 rotary microtome. After being deparaffinized, the sections = were=20 processed for immunohistochemistry. For floating sections, = perfused=20 brains were soaked in 30% sucrose overnight, frozen in = isopentane=20 cooled by dry ice, and cut on a cryostat into sections 30 = =E2=88=9Dm in=20 thickness. NSPCs were cultured on laminin-coated glass wells = and=20 were fixed in 4% PFA for 20 minutes at room temperature. = Cells and=20 brain sections were processed for immunohistochemistry using = the=20 following primary antibodies: rat monoclonal anti-BrdU (1:50 = dilution; Accurate Chemical, Westbury, New York, USA); = rabbit=20 polyclonal anti-SCF (1:100 dilution; Chemicon), and mouse = monoclonal=20 anti-nestin (1:400 dilution; Chemicon); mouse monoclonal = anti=E2=80=93glial=20 fibrillary acidic protein (anti-GFAP) (1:400 dilution;=20 Sigma-Aldrich, St. Louis, Missouri, USA), mouse monoclonal=20 anti=E2=80=93microtubule-associated proteins 2 (anti-MAP2) = (1:1,000=20 dilution; Sigma-Aldrich); rat monoclonal anti-CD13 (1:100 = dilution;=20 BD PharMingen); rabbit polyclonal anti=E2=80=93c-kit (1:100 = dilution; H-300;=20 Santa Cruz Biotechnology); mouse monoclonal = anti=E2=80=93=CE=B2-tubulin type III=20 (anti=E2=80=93TUJ-1) (1:2,000 dilution; Covance research = products, Berkeley,=20 California, USA); biotinylated lectin ricinus communis = agglutinin I=20 (RCA I) (1:1,000 dilution, Vector Laboratories, Burlingame,=20 California, USA); and rabbit polyclonal = anti=E2=80=93phosphorylated histone=20 H3 (anti=E2=80=93phospho-histone H3) (1:200 dilution; = Upstate Biotechnology,=20 Lake Placid, New York, USA). In order to prevent nonspecific = binding=20 of antibodies, cells or sections were treated with 5% normal = goat=20 serum in PBS with 0.1% BSA. The specificity of = immunolabeling was=20 verified in all experiments by controls in which the primary = antibody was omitted. Secondary antibodies tagged with Alexa = Fluor=20 488 and 594 (Molecular Probes, Eugene, Oregon, USA) were = used for=20 visualization at a dilution of 1:500 for each. For dual and = triple=20 staining, the primary antibodies used in one section were = from=20 different species and single-antibody staining was performed = to=20 ensure specificity of staining. Specimens were examined on = Zeiss LSM=20 510 confocal imaging system (Zeiss, Heidelberg, Germany) for = immunofluorescence. Individual optical sections (optical = depth, less=20 than 0.1 =E2=88=9Dm) were obtained for different fluorogens = and stacked=20 optical sections were merged using Maximum Projection = Software=20 (Zeiss). For quantitative immunocytochemical data, = Abercrombe=E2=80=99s=20 correction was used to approximate the number of cells with = positive=20 staining per examined area and a percentage per total cells = within=20 the area. Data were expressed as means =C2=B1 SEM.

BrdU labeling and intracerebral stereotactic = injections.=20 For labeling of neural progenitors in vivo, BrdU = (Roche) was=20 administrated intraperitoneally twice a day, at 8-hour = intervals,=20 for 14 days (50 mg/kg body weight). Twenty hours after the = last BrdU=20 injection, several animals were killed and their brains were = sectioned for BrdU immunostaining. Intracerebral injections = were=20 administered to the remaining animals. Stereotactic surgery = was=20 performed as described for the =E2=80=9Cfreeze=E2=80=9D = injury, and injections were=20 given at the position of 1 mm anterior to the bregma, 1.5 mm = lateral=20 to the midline, and 2.5 mm ventral to the dura. Mice = received a=20 suspension of rmSCF (3 =E2=88=9Dg in 3 =E2=88=9Dl of PBS; = R&D Systems) or PBS=20 through a Hamilton syringe. Each injection took 15 minutes. = Seven=20 days after the intracranial injection, the animals were = killed and=20 their brains were processed for BrdU immunohistochemical = analysis.=20 For ventricular injection of DiI=20 = (1,1=E2=80=B2-dioctadecyl-6,6=E2=80=B2-di(4-sulfophenyl)-3,3,3=E2=80=B2,3= =E2=80=B2-tetrametylindocarbocyanine;=20 Molecular Probes) or adenovirus expressing GFP, 2 =E2=88=9Dl = of 0.2 % DiI in=20 DMSO or 2 =E2=88=9Dl of replication-incompetent adenovirus = (108=20 plaque-forming units per microliter) (36) = was injected=20 0.5 mm posterior and 0.7 mm lateral to bregma, and 2 mm = below the=20 dura matter, into the lateral ventricle. Three days after=20 ventricular injection, SCF was injected into the left = hemisphere as=20 described above. Seven days after SCF injection, the brains = were=20 sectioned and analyzed for DiI or GFP distribution.

Results

SCF expression induced by =E2=80=9Cfreeze=E2=80=9D = injury in brain.=20 =E2=80=9CFreeze=E2=80=9D injuries were introduced = into the right frontal=20 lobe of mouse brain as illustrated in Figure 1A. Genes that = were=20 differentially expressed in brain tissue 5 days after injury = were=20 globally screened through SSH using contralateral uninjured = tissue=20 as a control. The differential expression of the isolated = clones was=20 verified using custom microarray. More than 300 clones, = representing=20 196 unique genes, were identified. The microarray membranes, = which=20 contained the individual clones from the SSH library in = duplicate,=20 were hybridized with cDNA from injured frontal lobe or with = cDNAs=20 from the uninjured contralateral side. The differentially = expressed=20 clones were subsequently sequenced and two of them were = identified=20 as SCF (Figure 1B). The = injury-mediated=20 induction of SCF mRNA expression was further = confirmed by=20 quantitative real-time RT-PCR, revealing an increase of more = than=20 twofold in SCF transcripts in the injured tissue = (Figure 1C).

We next investigated whether SCF protein, in parallel to=20 SCF mRNA, was also increased in the injured brain. = Western=20 blot confirmed the presence of two SCF bands corresponding = to the=20 transmembrane and cleaved soluble forms of SCF (33 and 19 = kDa,=20 respectively; Figure 1D). Compared = with that=20 of the corresponding forebrain tissue of uninjured mice, SCF = protein=20 levels increased as early as day 1 after injury, with the = soluble=20 form reaching maximum induction by day 5 and the = membrane-bound form=20 peaking 7 days after injury. The total amount of SCF also = peaked at=20 day 7 and then declined by day 12 after injury (Figure 1E). Finally, = we=20 analyzed the distribution of SCF protein in the injured = forebrain.=20 SCF immunohistochemical staining was barely detectable in = the=20 uninjured control forebrains and was only weakly detectable = in=20 cortical layers I and II (Figure 2, A and C). = In=20 contrast, prominent staining was seen in cells in layers II, = III,=20 IV, and V of the injured neocortex, with extension up to the = corpus=20 callosum (Figure 2, B=20 and D). SCF staining was also observed in the SVZ of injured = brain=20 but was not seen in the control brain (Figure 2, E and F). = In the=20 contralateral, uninjured hemisphere, we observed some SCF = staining=20 in the SVZ and neocortex that was adjacent to the injured = side. We=20 did not, however, observe increased SCF expression in the = areas of=20 the contralateral uninjured brain at a distance from the = site of=20 injury (data not shown). At a microscopic level it was = apparent that=20 the highest level of SCF expression occurred in cells = immediately=20 adjacent to the site of direct injury, with a slow drop off = in the=20 level of expression as the distance increased away from the = injury=20 site (Figure 2, G and=20 H). We initially expected that the majority of the SCF would = originate from inflammatory cells, specifically blood-borne=20 monocytes and or microglia; however, only a minority of the=20 SCF-expressing cells within the injured brains showed = positive=20 staining for microglia and monocytes markers, including = lectin-RCA=20 I, F4/80, and CD13 (Figure 3, G and H, = and data not=20 shown). Likewise, the majority of GFAP-positive cells did = not=20 express SCF (Figure 3, E and F). = Instead=20 there was intense SCF staining of the neuropil, suggesting a = predominantly neuronal expression of SCF. = Immunohistochemical=20 studies confirmed that the majority of SCF-expressing cells = were=20 positive for TUJ-1 and/or MAP2, consistent with neuronal SCF = expression (Figure 3,=20 A=E2=80=93D).

Figure=20 2

Distribution of SCF in the injured forebrain. = (A_G)=20 Sections were immunostained with an Ab against SCF and were=20 visualized with DAB (brown). Counterstaining was done with=20 hematoxylin (blue). (A) In the normal cortex, = SCF-positive=20 cells exist mainly in the surface of the cortex, in layers I = and II.=20 (B) In the =E2=80=9Cfreeze-injured=E2=80=9D = forebrain, the distribution of=20 SCF-staining cells include the whole depth of cortex, with=20 intensively SCF-positive cells in the layers III, IV. = (C and=20 D) Magnified images of the black boxes in A = and=20 B, respectively. Abundant SCF-staining cells were = observed in=20 the injured area indicated by arrows in D. (E = and=20 F) SCF expression was also present in the SVZ of = injured=20 brain (F) but was not detectable in the control SVZ=20 (E). (G) Large numbers of SCF-staining cells = were=20 present around the injury (boxes I and II), while the = SCF-positive=20 cells decreased with increasing distance away from the site = of=20 injury (boxes III and IV). Bottom row, higher-magnification = views=20 (=E2=88=9E200) of each section as indicated by the arrows. = Boxes I_IV are=20 schematic representations of the areas used to quantify cell = numbers. Scale bar (shown in A): A and = B, 200=20 =E2=88=9Dm; C_F, 50 =E2=88=9Dm; G, main = image, 250 =E2=88=9Dm, and=20 higher magnifications, 70 =E2=88=9Dm. Str, striatum. = (H)=20 Quantification of SCF positive cells in boxes I_IV of = G. The=20 SCF-positive cells are presented as a percentage of the = total cell=20 number for each section. Comparisons are corrected for = surface area=20 and total cells in the section. Statistical differences were = determined by comparison of boxes III and IV with box I = (*P=20 < 0.05).

Figure=20 3

Characterization of SCF-positive cells in the cortex 7 = days after=20 injury. (A_H) Sections were double = immunostained for=20 TUJ-1 and SCF (A and B), MAP2 and SCF = (C and=20 D), GFAP and SCF (E and F), or lectin = RCA I and=20 SCF (G and H). White boxes in A, = C,=20 E, and G are magnified in B, D,=20 F, and H, respectively. The enlarged images = show=20 double staining for TUJ-1 and SCF or MAP2 and SCF (arrows), = and=20 GFAP, RCA I or SCF single-positive cells (arrowheads). Scale = bar=20 (shown in A): A, C, E, and = G , 50=20 =E2=88=9Dm; B, D, and H, 16 =E2=88=9Dm; = F, 18=20 =E2=88=9Dm.

Expression of c-kit on the NSPCs and responsiveness = to SCF.=20 Human fetal and mouse NSPC populations from = embryonic day=20 14.5 were expanded in serum-free media containing bFGF and = EGF.=20 These undifferentiated brain-derived NSPCs grew in = neurospheres=20 under our experimental conditions and avidly expressed = nestin, a=20 marker for NSPCs. In contrast, the cells showed negative = staining=20 for endothelia and mesenchymal differentiation markers (data = not=20 shown). The true NSPC-like nature of our cells was confirmed = by=20 exposure of the cells to differentiating conditions, which = resulted=20 in expression of TUJ-1, GFAP, and CNPase in cells with = morphologies=20 consistent with those of neuronal, astrocytic, and = oligodendrocytic=20 lineages, respectively (data not shown).

To more directly address the potential effect of SCF on = neural=20 progenitor migration, we examined c-kit (SCF receptor) = expression=20 and activation on the mouse NSPCs as well as human NSPCs.=20 Semiquantitative RT-PCR revealed abundant c-kit mRNA = in the=20 cultured mouse and human NSPCs (Figure 4A).=20 Immunohistochemistry confirmed the expression of c-kit = receptor in=20 the cultured mouse and human NSPCs (Figure 4, B and C). = In=20 addition, we could readily identify a subpopulation of=20 nestin-positive cells in the SVZ that were c-kit positive = (Figure 4, D and E), = indicating=20 c-kit expression on adult neural progenitors in vivo.

Figure=20 4

Expression of c-kit in NSPCs in vitro and in vivo. = (A)=20 Expression of c-kit mRNA in human NSPCs (hNSPC) and = mouse=20 NSPCs (mNSPC), as measured by semiquantitative RT-PCR. = (B and=20 C) Immunofluorescence analysis for c-kit and nestin = in mouse=20 (B) and human (C) NSPCs. (D and = E)=20 Expression of c-kit in NSPCs in vivo. (D) Double = staining of=20 mouse brain by c-kit and nestin revealed c-kit expression in = nestin-positive cells (arrows) in the SVZ. (E) = Representative=20 orthogonal images of colocalization of c-kit and nestin in = vivo.=20 Upper, main, and left panels show views of the xz, = xy,=20 and yz planes, respectively. Lines represent = coordinates in=20 each plane. x axis, 24.4 =E2=88=9Dm; y axis, = 24.4 =E2=88=9Dm; z=20 axis, 6.7 =E2=88=9Dm; optical section thickness, 0.02 = =E2=88=9Dm. Scale bar (shown=20 in B): B_D, 20 =E2=88=9Dm.

The binding of SCF to its receptor, c-kit, is known to = induce=20 c-kit autophosphorylation on tyrosine residues, resulting in = activation of various downstream signaling pathways. In = order to=20 evaluate whether the c-kit receptor was functional on neural = progenitor cells, we stimulated the NSPCs with 100 ng/ml of = rmSCF=20 followed by immunoprecipitation of cellular proteins using a = c-kit=20 Ab. Immunoprecipitated proteins were then immunoblotted with = a=20 phosphotyrosine Ab followed by reprobing of the blot with a = c-kit=20 Ab, as described in Methods. The results demonstrated that = c-kit=20 proteins on NSPCs were strongly phosphorylated after SCF=20 stimulation. Moreover, pretreatment of mouse NSPCs with a = specific=20 c-kit=E2=80=93blocking Ab (ACK 45) markedly decreased c-kit=20 autophosphorylation on mouse NSPCs (Figure 5A). These = data=20 demonstrate that the c-kit receptor is biologically active, = as=20 determined by tyrosine kinase activity, on neural progenitor = cells.

Figure=20 5

SCF/c-kit pathway is involved in injury-induced NSPC = migration.=20 (A) SCF-induced c-kit tyrosine phosphorylation = (Tyr-P) was=20 detected by immunoprecipitation (IP) and immunoblot (IB). = The=20 120-kDa and 140-kDa bands represent the c-kit proteins in = the human=20 and mouse NSPCs. Lane 1, SCF treatment; lane 2, untreated = control;=20 lane 3, pretreatment of NSPCs with ACK45 c-kit_blocking Ab = before=20 SCF treatment. (B) Tissue lysates from injured and = normal=20 mouse forebrain were used to stimulate migration of mouse = NSPCs=20 (with or without pretreatment with ACK45 blocking Ab) in the = Boyden=20 chamber migration assay. Relative fluorescence units (RFU)=20 correlated with the number of migrated cells. NSPC migration = was=20 significantly induced by injured brain lysates compared with = normal=20 brain lysates (*P < 0.05). The chemotactic effect = of=20 injured brain lysates was nearly completely abolished when = NSPCs=20 were pretreated with the c-kit_blocking Ab (*P < = 0.05).=20 Error bars represent SEM. These are representative = experiments and=20 similar results were obtained from at least three = independent=20 experiments.

Chemoattractant effects of SCF on NSPC migration in = vitro.=20 We next asked if the SCF upregulation in the injured = brain=20 was functionally relevant for neural progenitor migration. = We=20 established a Boyden chamber=E2=80=93based migration assay = to quantitatively=20 evaluate NSPC migration in vitro. Lysates from injured = brains=20 induced significantly more cell migration than did lysates = from=20 normal brains. Additionally, the increased cell migration = seen after=20 exposure of NSPCs to injured brain lysates could be = abolished by=20 pretreatment of the NSPCs with the ACK45 = c-kit=E2=80=93blocking Ab (Figure=20 5B). This = migration=20 inhibition did not occur after pretreatment with a control = rat IgG=20 (data not shown).

To directly evaluate the chemoattractant effects of SCF = on neural=20 stem cell migration, we used various concentrations = (5=E2=80=93500 ng/ml) of=20 purified rmSCF to induce NSPC migration in the Boyden = chamber=20 migration assay. We observed that c-kit mRNA was not = detectable in=20 the NIH 3T3 cells by RT-PCR (Figure 4A), and = therefore we=20 included NIH 3T3 cells in the Boyden chamber assay as a = negative=20 control. In the presence of 1% serum, NSPCs and NIH 3T3 = cells=20 demonstrated the same minimal nonspecific migratory = response. The=20 response to rmSCF, however, was quite different between = these cell=20 types; rmSCF did not induce migration of NIH 3T3 cells = regardless of=20 the concentration used. In contrast, rmSCF resulted a = significant=20 chemoattractant effect on NSPCs in a dose-dependent manner = (Figure=20 6). = Statistically=20 significant increases in cell migration were seen at SCF=20 concentrations as low as 5 ng/ml for mouse NSPCs and at 10 = ng/ml for=20 human NSPCs. This experiment was repeated three times and = similar=20 results were obtained. These data demonstrate that SCF = regulates=20 NSPC migration in vitro.

Figure=20 6

Effects of rmSCF on NSPC migration in the Boyden chamber=20 migration assay. Mouse and human NSPC migration was = stimulated in a=20 dose-dependent manner by soluble rmSCF. Error bars represent = SEM.=20 *P < 0.05, and **P < 0.001, = respectively.=20 n =3D 3. This is a representative experiment; similar = results=20 were obtained from three independent experiments.

Chemoattractant effects of SCF on neural stem cell=20 migration in vivo. To test the effects of SCF on = endogenous=20 progenitor cell migration in vivo, we prelabeled endogenous = dividing=20 NSPCs with BrdU in SCID mice. Twenty hours after the last = BrdU=20 injection, several brains were processed for BrdU=20 immunohistochemistry analysis as a day-0 control. The = remaining=20 animals were divided into two groups: those with = intracerebral=20 injection of rmSCF and those injected with the vehicle = control=20 (PBS). Treated animals were euthanized on day 7 after SCF or = PBS=20 administration. The brain sections were then evaluated for=20 expression of BrdU, nestin, and phospho-histone H3 by=20 immunohistochemistry and fluorescence confocal microscopy. = The=20 SCF-treated group had significant numbers of BrdU-positive = cells in=20 the SCF-injected area compared with both the contralateral = side of=20 the same brain and the PBS-injected brains (Figure 7, A and B). = There were=20 approximately twice the number of BrdU-positive cells in the = SCF-injected area (1.5-mm2 section) compared with the = contralateral=20 side and 2.3 times the number of cells in the injected area = in=20 SCF-treated animals compared with PBS-treated animals = (P <=20 0.01). In contrast, in the day-0 group animals, the majority = of=20 dividing cells were distributed within the SVZ, with only a = few=20 cells widely dispersed within the corpus callosum, cerebral = cortex,=20 and striatum (Figure 7D). Moreover, = BrdU=20 incorporation colocalized with the cell proliferation marker = phospho-histone H3, demonstrating that BrdU incorporation = was a=20 function of cell division rather than DNA damage (Figure 7, = D=E2=80=93F). The majority of=20 BrdU-positive cells expressed nestin, indicating that they = were=20 NSPCs (Figure 7, G=20 and H).

Figure=20 7

SCF-stimulated progenitor cell migration in vivo. = (A)=20 Quantification of BrdU-labeled cells in the injected areas = (1.5 mm2)=20 and in the contralateral side of the brain. Significantly = more=20 BrdU-labeled cells were seen in the SCF-injected area than = in the=20 PBS-injected area or in the contralateral side of the = SCF-injected=20 brain (P < 0.001; n =3D 6). inj., injection = side;=20 contra., side contralateral to SCF injection. (B)=20 Representative images of BrdU staining in the cortical = areas.=20 (C) Schematic diagram of the brain used to examine = the=20 response of NPSC to rmSCF is shown. The blue arrow indicates = the=20 intracerebral injection track (as described in Methods). = (D)=20 Normal distribution of BrdU-positive cells. These cells were = detected mainly in the SVZ prior to SCF injection and were = also=20 positive for phospho-histone H3 staining (p-H3). (E = and=20 F) Immunohistochemistry of SCF-injected brain with = BrdU and=20 phospho-histone H3 antibodies in LVZ (E) and = SCF-injected=20 cortex (F). (G) Nestin expression of = BrdU-positive=20 cells in the SVZ. (H) Three-dimensional digital image = of the=20 cells indicated by the arrowhead in G is shown. = Upper, main,=20 and right panels show views of the xz, xy, and = yz planes, respectively. Lines represent coordinates = in each=20 plane. x axis, 23.2 =E2=88=9Dm; y axis, 23.2 = =E2=88=9Dm; z=20 axis, 11 =E2=88=9Dm; optical section thickness, 1.1 = =E2=88=9Dm. Scale bar (shown in=20 B): B, 100 =E2=88=9Dm; D, 64 = =E2=88=9Dm; E and F,=20 32 =E2=88=9Dm; G, 12 =E2=88=9Dm.

To clearly demonstrate that the BrdU-positive stem cells = were SVZ=20 stem cells that had migrated to the SCF injection site, = rather than=20 representing the proliferation of a local population of stem = cells,=20 we injected DiI fluorescent dye or adenovirus expressing GFP = into=20 the cerebral ventricle of the mouse. As has been previously=20 demonstrated (37), = ventricular=20 injection of DiI or GFP adenovirus enables one to track the = progeny=20 of NSPCs in the SVZ region. The ventricular injection of DiI = or=20 adenovirus expressing GFP was given prior to SCF = administration. The=20 injected DiI was restricted to areas near ventricular zones = (Figure=20 8A), = eliminating the=20 possibility of diffusion of free dye into other areas of the = brain,=20 thereby ensuring the labeling of cells that were immediately = adjacent to the ventricle (Figure 8B). After = injection of=20 SCF, significant numbers of DiI-labeled cells were found = near the=20 injection site of SCF. In contrast, few DiI-labeled cells = were=20 detected in the contralateral cortex of SCF-injected animals = or near=20 the injection site in animals injected with PBS (Figure 8, C and D). = Similarly,=20 GFP expression was initially confined to cells immediately = adjacent=20 to the ventricular system after injection of GFP-expressing=20 adenoviral vectors into the ventricles (Figure 8E). After SCF = injection, however, a large number of GFP-positive cells had = migrated into the SCF-injected cortex (Figure 8, F and G). = Taken=20 together, these ventricular labeling experiments clearly = show that=20 SVZ stem cells migrate into the SCF-injected cortex.

Figure=20 8

SCF-stimulated progenitor cell migration in vivo. = (A and=20 B) Whole-brain image of DiI labeling after = ventricular=20 injection of DiI. DiI-labeled cells (red) were only detected = in the=20 lining of ventricular zone. Boxed regions in A are = shown at=20 higher magnification in B. DiI staining was confined = to the=20 nuclei and process of cells. (C and D) = Representative=20 images of control hemisphere (C) and SCF-injected = hemisphere=20 (C). Note the greater number of DiI-positive cells in = the SCF=20 injection side. (E) Whole-brain image of GFP-positive = cells=20 (green) in the brain. 3V, third ventricle. (F and = G)=20 Representative images of control hemisphere (F) and=20 SCF-injected hemisphere (G). Scale bar (shown in = B):=20 A and E, 100 =E2=88=9Dm; = B_D,F, and=20 G, 20 =E2=88=9Dm.

Discussion

NSPCs have notable migratory abilities within the CNS = under=20 physiological and pathological conditions such as during = embryonic=20 nervous system development, adult neurogenesis, and injury = response.=20 Although there is a growing body of literature regarding the = mechanisms responsible for NSPC migration during CNS = development,=20 little is known of the mechanisms that are operative in = mediating=20 NSPC migration during times of injury. Understanding of the = signals=20 responsible for NSPC migration to sites of brain and spinal = cord=20 injury could prove crucial for the development of strategies = for=20 NSPC-mediated repair of such damage.

To begin to elucidate the signals involved in NSPC = migration to=20 sites of CNS injury, we constructed a =E2=80=9Csubtraction = library=E2=80=9D of=20 injured brain tissue compared with control brain tissue. We=20 identified more than 300 clones representing 196 unique = genes that=20 are differentially expressed in the injured tissue. In order = to=20 prioritize clones that might be most interesting to evaluate = first,=20 we made the assumption that a neural progenitor = chemoattractant=20 would likely be a membrane-bound and/or secreted protein = with the=20 ability to diffuse within the extracellular space of the = brain,=20 given the pronounced effect of injury on NSPC migration at=20 relatively large distances. Thus, we performed a = bioinformatics=20 screen of our clones, looking for genes that contained a = predicted=20 signal peptide. One of the most prominent clones represented = in our=20 subtraction and bioinformatics screen was that encoding SCF. = Our=20 data demonstrate that SCF mRNA is overexpressed in = injured=20 brain compared with normal brain by SSH analysis, custom = cDNA=20 microarray analysis, and real-time quantitative RT-PCR.=20 Correspondingly, SCF protein is also induced by injury.

A single SCF gene that maps to 12q22-24 encodes = two=20 different isoforms of the protein (38, = 39). A = longer,=20 248=E2=80=93amino acid SCF isoform contains a proteolytic = cleavage site at=20 Ala 165 that, after cleavage, results in loss of the = transmembrane=20 region and a soluble truncated protein (40, = 41). = Both SCF=20 isoforms bind and activate the c-kit receptor, although = their=20 effects on c-kit signaling may be subtly different secondary = to=20 differences in receptor internalization (42).=20 Specifically, the soluble c-kit/SCF complex is rapidly = internalized=20 and degraded, resulting in transient tyrosine kinase = activation of=20 c-kit, whereas the membrane-bound SCF appears to prevent = receptor=20 internalization, resulting in more persistent receptor = tyrosine=20 phosphorylation (43=E2=80=9345).=20 Loss-of-function mutations in SCF or c-kit have = demonstrated=20 the importance of the SCF/c-kit pathway in hematopoiesis,=20 gametogenesis, and melanogenesis. In contrast, activating = mutations=20 in c-kit mediate transformation of hematopoietic stem cells, = mast=20 cells, and gastrointestinal stromal cells (46=E2=80=9351). = Recent=20 reports have indicated that the SCF/c-kit pathway is = activated=20 during injury and plays a protective role in selected injury = models=20 of non-neural tissue (52, = 53). = In addition=20 to these effects on cellular proliferation, differentiation, = survival, and transformation, SCF can also induce migration = of=20 hematopoietic stem cells, melanoblasts, and mast cells = (54=E2=80=9360).

Our data are consistent with a previous report = demonstrating that=20 SCF expression is induced by brain injury in neurons = surrounding the=20 injury, as assessed by in situ hybridization and=20 immunohistochemistry (61).=20 Additionally, a recent study has shown increased SCF levels = in the=20 medium from cerebral cortical cultures after hypoxic insult, = suggesting that SCF is also induced in other types of neural = injury=20 such as cerebral ischemia (62). = We have now=20 extended these observations by demonstrating not only that = SCF is=20 overexpressed in neurons after traumatic injury in vivo but = also=20 that recombinant SCF can directly induce neural stem cell = migration=20 in vitro and in vivo. These data, therefore, suggest a role = for SCF=20 in mediating NSPC migration to areas of acute brain injury. = Of=20 interest is a previous report suggesting that the brain=20 predominantly produces the soluble form of SCF (63). = Our Western=20 blot data, however, clearly demonstrate that although the = soluble=20 form of SCF is predominant, membrane-bound SCF is also = increased in=20 the traumatized brain. Given the differences in the degree = and=20 kinetics of c-kit activation between the two different SCF = isoforms,=20 it is tempting to speculate that the production of both the = soluble=20 and membrane forms of the cytokine more readily allows for = the=20 establishment of a chemotaxis gradient toward the site of=20 injury.

In addition to demonstrating the direct migratory effects = of SCF=20 on NSPCs, we have also demonstrated the presence of c-kit on = NSPCs,=20 the resultant tyrosine phosphorylation of the receptor after = exposure to SCF, and the presence of activated receptor on=20 progenitor cells in the SVZ of the lateral ventricle and = subgranular=20 zone of the hippocampal dentate gyrus. These data are = consistent=20 with those of Jin and co-workers, who demonstrated c-kit = expression=20 in neuroproliferative zones in the adult mammalian brain = after=20 ischemic injury (62). = The signal=20 transduction pathways operative in NSPC migration after = c-kit=20 activation remain to be fully elucidated, although recent = evidence=20 suggests that c-kit=E2=80=93mediated mast cell migration is = dependent on=20 phosphoinositide-3 kinase (PI-3Kinase) signaling (64). = Recent work=20 has demonstrated that mouse NSPCs heterozygous for = phosphatase and=20 tensin homolog deleted on chromosome 10 (PTEN) have = increased=20 migratory properties and that conditional PTEN knockout mice = have=20 disorganization of brain neuronal architecture, probably due = to=20 precursor migratory errors. These data are consistent with a = central=20 role for PI-3Kinase in NSPC migration, as PTEN is a negative = regulator of components of PI-3Kinase signaling (64, = 65). = Finally,=20 recent data suggest a role for Slug, a member of the Snail=20 zinc-finger family of transcription factors, in SCF-mediated = migration of hematopoietic and gamete stem cells (66). = Although=20 further studies are clearly needed to precisely define the = role of=20 PI-3Kinase and Slug in c-kit=E2=80=93induced NSPC migration, = our data=20 clearly demonstrate that the SCF/c-kit pathway is activated = within=20 the brain after both traumatic and ischemic injury and = contributes=20 to NSPC migration to those sites of injury.

Although both SCF expression in neurons and c-kit = activation on=20 NSPCs after injury have been described previously, this is = the first=20 report to our knowledge that has made the functional = connection=20 between neuronally expressed SCF and induction of NSPC = migration to=20 the site of injury. Given the known migratory effects of SCF = on=20 other types of stem cell populations, it is perhaps not = entirely=20 surprising that SCF has such effects on NSPCs. We do not at = this=20 time understand the significance of injury-induced = SCF-mediated=20 neural stem cell migration to sites of injury, although it = is=20 tempting to speculate that recruitment of NSPCs is part of = an injury=20 repair process. Whether SCF/c-kit=E2=80=93mediated = recruitment of NSPCs to=20 sites of injury actually accomplishes this goal, what other=20 molecules are operative in this process, and whether SCF is = playing=20 an additional role in injury-induced neurogenesis and stem = cell=20 differentiation within the area of injury are important = questions=20 that will require additional study. Irrespective of the=20 physiological role of SCF in the brain, the elucidation of = SCF as a=20 potent NSPC migratory factor opens up an opportunity to = utilize=20 recombinant or genetic vector=E2=80=93derived SCF as a = chemotactic agent to=20 induce NSPC recruitment to specific areas of the CNS for = cell-based=20 therapeutic interventions.

Acknowledgments

The authors thank Jim Nagle and Deborah Kauffman of the = National=20 Institute of Neurological Disorders and Stroke sequencing = facility=20 for technical support in DNA sequencing.

Footnotes

Nonstandard abbreviations used: glial fibrillary = acidic=20 protein (GFAP); microtubule-associated protein 2 (MAP2); = neural=20 stem/progenitor cell (NSPC); paraformaldehyde (PFA);=20 phosphoinositide-3 kinase (PI-3Kinase); recombinant mouse = SCF=20 (rmSCF); ricinus communis agglutinin I (RCA I); rostral = migratory=20 stream (RMS); stem cell factor (SCF); subtractive = suppression=20 hybridization (SSH); subventricular zone (SVZ); = =CE=B2-tubulin type III=20 (TUJ-1).

Conflict of interest: The authors have declared = that no=20 conflict of interest exists.

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