Experimental Neurology

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1 Experimental Neurology 221 (2010) Contents lists available at ScienceDirect Experimental Neurology journal homepage: Bone morphogenetic proteins mediate cellular response and, together with Noggin, regulate astrocyte differentiation after spinal cord injury Qi Xiao a, Yang Du a, Wutian Wu a,c, Henry K. Yip a,b,c, a Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China b Research Center of Heart, Brain, Hormone and Healthy Aging Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China c State Key Laboratory of Brain and Cognitive Sciences, The University of Hong Kong, Pokfulam, Hong Kong SAR, China article info abstract Article history: Received 4 August 2009 Revised 31 October 2009 Accepted 1 December 2009 Available online 11 December 2009 Keywords: BMP psmad Noggin pstat Spinal cord injury Neural stem cell Differentiation Astrogenesis Bone morphogenetic proteins (BMPs) play a critical role in regulating cell fate determination during central nervous system (CNS) development. In light of recent findings that BMP-2/4/7 expressions are upregulated after spinal cord injury, we hypothesized that the BMP signaling pathway is important in regulating cellular composition in the injured spinal cord. We found that BMP expressions were upregulated in neural stem cells (NSCs), neurons, oligodendrocytes and microglia/macrophages. Increased expression levels of psmad1/ 5/8 (downstream molecules of BMP) were detected in neurons, NSCs, astrocytes, oligodendrocytes and oligodendroglial progenitor cells (OPCs). Active astrocytes which form the astroglial scar were probably derived from NSCs, OPCs and resident astrocytes. Since quiescent NSCs in the normal adult spinal cord will proliferate and differentiate actively into neural cells after traumatic injury, we proposed that BMPs can regulate cellular components by controlling NSC differentiation. Neurosphere culture from adult mouse spinal cord showed that BMP-4 promoted astrocyte differentiation from NSCs while suppressing production of neurons and oligodendrocytes. Conversely, inhibition of BMP-4 by Noggin notably decreased the ratio of astrocyte to neuron numbers. However, intrathecal administration of Noggin in the injured spinal cord failed to attenuate glial fibrillar acidic protein (GFAP) expression even though it effectively reduced psmad expression. Noggin treatment did not block phosphorylation of Stat3 and the induction of GFAP in the injured spinal cord, suggesting that in addition to the BMP/Smad pathway, the JAK/STAT pathway may also be involved in the regulation of GFAP expression after spinal cord injury Elsevier Inc. All rights reserved. Introduction In contrast to injury to the peripheral nervous system (PNS), which possesses a favorable environment for both short-range remodeling and long-distance axon regrowth after injury, adult CNS injury often results in persistent damage because the mature axons cannot regenerate in a glial environment containing inhibitory molecules (Yiu and He, 2006). After CNS injury, reactive astrocytes increase in number, form a glial scar and secrete large amounts of chondoitin sulfate proteoglycans (CSPGs), which prevent axon repair (Silver and Miller, 2004). In addition, myelin-associated inhibitors produced by oligodendrocytes and myelin debris in the CNS, such as Nogo, ephrin B3, myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp) and transmembrane semaphorin 4D (Sema4D/ CD100), create a growth inhibitory environment in the injured CNS and trigger growth cone collapse (Mukhopadhyay et al., 1994; Wang et al., 2002; Moreau-Fauvarque et al., 2003; Benson et al., 2005). Corresponding author. Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China. Fax: address: hkfyip@hku.hk (H.K. Yip). Since injured adult spinal cord may recapitulate important developmental events during the regenerative process, morphogens important in the regulation of cell proliferation and differentiation in the developing nervous system may also exert pleiotropic effects on multiple cellular constituents of the injured adult nervous tissues (Chen et al., 2005; Hall and Miller, 2004; Harvey et al., 2005). Bone morphogenetic proteins (BMPs), which belong to the TGF-β superfamily, play an important role in regulating the development of the CNS (Helm et al., 2000). Extracellular antagonists such as Noggin can suppress the BMP signaling pathway by binding BMP ligands to prevent their association with BMP receptors (Liu and Niswander, 2005). In vitro experiments have demonstrated that BMPs mediate differentiation and maturation of astrocytes from adult mouse spinal cord-derived progenitor cells while suppressing both neuronal and oligodendroglial lineage (Setoguchi et al., 2004). In addition, BMPs promote production of astrocytes from OPCs at the expense of oligodendrocytes. Mabie et al. (1997) found that OPCs from postnatal rat cortex differentiated into mainly astrocytes instead of oligodendrocytes after BMP-2 stimulation. Since BMPs are involved in promoting astrogliogenesis at the late stage of CNS development, and their expressions are significantly /$ see front matter 2009 Elsevier Inc. All rights reserved. doi: /j.expneurol

2 354 Q. Xiao et al. / Experimental Neurology 221 (2010) upregulated after injury (Setoguchi et al., 2004, 2001), and they increase reactive astrocyte production to form glial scars which restrict axon regrowth (Fuller et al., 2007), BMPs are considered as neurite outgrowth inhibitors. Treating cultured astrocytes with BMP- 4 and BMP-7 increased CSPG protein expression, which was consistent with the view that BMPs increase gliogenesis and glial scar formation at the lesion site (Fuller et al., 2007). In addition, BMPs can take part in inhibiting oligodendrocyte specification and differentiation to promote astrocyte proliferation after injury. Treatment of OPCs with BMP significantly increased Id2 and Id4 expressions, which inhibited OPC differentiation into mature oligodendrocytes through their interaction with Olig1 and Olig2 (Samanta and Kessler, 2004). On the other hand, blocking the BMP signaling pathway by overexpression of Noggin in neural precursor cells transplanted into the spinal cord lesion site induced neuronal and oligodendroglial lineage commitment and promoted functional recovery of paraplegic mice (Setoguchi et al., 2004). There have been numerous in vitro studies on the effects of BMPs on cell proliferation and differentiation in many different cell types, including NSCs, OPCs, oligodendrocytes and astrocytes. However, the expression patterns of BMPs and the effects of BMPs on the regulation of cell components in the injured CNS in vivo have not been determined. In this study, we characterized the temporal expressions of BMP-2/-4/-7 and the downstream signaling molecules psmad1/5/ 8 in adult mice spinal cords following contusive injury. We examined the effect of Noggin infusion on the expression level of GFAP at the lesion site. In addition, we assessed the effects of BMPs and Noggin on the differentiation of NSCs derived from adult spinal cord. These findings provide an understanding of how BMP can regulate cell proliferation and differentiation in response to CNS injury and provide the basis of manipulation of adult spinal NSCs to repair spinal cord injury. Materials and methods Experimental spinal cord injury in mice Adult (6 8 weeks) female C57/BL6n mice (from the Laboratory Animal Unit at the University of Hong Kong) were used in this study. The animals were deeply anesthetized with intraperitoneal injection of ketamine (80 mg/kg) and xylazine (8 mg/kg). A laminectomy was performed to expose the dorsal portion of the spinal cord corresponding to thoracic level T8 and T9. Contusive injury was induced using a standard weight-drop instrument (New York University Impactor RUK-106). The 3 g impactor rod was dropped from a height of 25.0 mm onto the exposed spinal cord, and then lifted off the spinal cord immediately after the impact. In the second series of experiments, an osmotic minipump (Alzet 1007D: 1 week infusion; volume: 100 μl; rate of infusion: 0.5 μl/h; Alzet Osmotic Pumps Company, USA) filled with recombinant mouse Noggin (15 ng/kg/day, R&D Systems) or vehicle control diluent (0.01 M phosphate-buffered saline [PBS] +1% bovine serum albumin [BSA]) was placed under the skin of the mouse's back after the contusive injury. A silastic tube connected to the minipump was inserted at the lesion site under the dura of the spinal cord. The tubing was secured to the muscle of the back with a suture just caudal to the site of laminectomy. The overlying muscle and skin were sutured. All surgical procedures and post-operative care were performed in accordance with National Institutes of Health guidelines and were approved by the Committee on the Use of Live Animals in Teaching and Research, Faculty of Medicine, The University of Hong Kong. QT-PCR analysis Mice were killed by an overdose of pentobarbital sodium (100 mg/ kg, i.p.) at 12 h and 1, 2, 4 and 8 days after injury (n=3 at each time point). T8 T9 spinal cord segments were dissected and homogenized in 1 ml TRIZOL Reagent (Gibco, Life Technologies). Total RNA was further isolated by chloroform (0.2 ml chloroform per 1 ml TRIZOL Reagent), precipitated in 0.5 ml isopropyl alcohol per 1 ml TRIZOL Reagent, dried for 10 min and dissolved in 30 μl DEPC water. RNA quantity was assessed through spectrophotometric analysis by GeneQuant II RNA/DNA Calculator (Pharmacia Biotech). Using a commercially available kit (SuperScripts First-Strand Synthesis System, Invitrogen), 2 μg total RNA was reverse transcribed to cdna used as the template for quantitative polymerase chain reaction (QT- PCR). QT-PCR analysis was carried out using: BMP-2 primers (forward: 5 -CCAGGTTAGTGAATCAGAACAC-3 and reverse: 5 -TCATCTTGGTGCAAAGACCTGC-3 ), BMP-4 primers (forward: 5 -ATTGGCTCCCAAGAATCATGG-3 and reverse: 5 -CGTGATGGAAACTCCTCACAGT), BMP-7 primers (forward: 5 -CGATTTCAGCCTGGACAACG-3 and reverse: 5 -CCTGGGTACTGAAGACGG-3 ) and β-actin primers (forward: 5 -AGCCATGTACGTAGCCATCC-3 and reverse: 5 -CTCTCAGCTGTGGTGGTGAA-3 ). β-actin was used as a control for equal amounts of template cdna in the reaction. The following were mixed together and loaded in a 96-well plate: 1.5 μl cdna, 1 μl forward primer (10 mm), 1 μl reverse primer (10 mm), 12.5 μl SYBR Green qpcr SuperMix (Invitrogen) and 6.5 μl DEPC water. QT-PCR was performed with an initial denaturation of the mixture for 10 min at 95 C, followed by 40 cycles of 30 s denaturation at 95 C, 30 s of annealing at 60 C and 1 min elongation at 72 C. SYBR green dye was used to produce the fluorescent signal that would be detected at the annealing phase and recorded by the sequence detector program. Two replicates were run for each cdna sample with BMP and β-actin primers in separate wells and the average data were recorded. PCR-amplified products were subjected to electrophoresis on a 1.0% agarose gel, stained with ethidium bromide, and visualized under ultraviolet light (White/Ultraviolet Transilluminator, Ultra Violet Products). Western blot analysis Animals that received no Noggin treatment were sacrificed at 12 h and 1, 2, 4 and 8 days after injury. Animals from the Noggin treatment group were sacrificed at 4 and 8 days after injury. T8 T9 spinal cord samples (n =3 at each time point) were taken from the normal uninjured control group, from the spinal cord injury group without any treatment and from the spinal cord injury groups treated with either Noggin or vehicle control. Spinal cord samples were homogenized in 100 μl lysis buffer with protease inhibitor (Roche; 1:100) and dithiothreitol (DTT) (1 mg/l; 1:500). The lysate was sonicated (three 3 s) and centrifuged at 12,000 g for 25 min at 4 C to separate the protein into the supernatant. Protein concentrations were measured using a protein assay kit (Bio-Rad) and Labsystems Multiskan MS Plate Reader (Analytical Instruments, LLC), and then 5 sample buffer was added, followed by heating the mixture for 8 min at 90 C. Equal amounts of protein extracts from each sample (30 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) on 10% polyacrylamide gel. Peptides were then electrophoretically transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore Corporation) for 3 h at 400 ma. Nonspecific binding was blocked by incubation for 1 h at room temperature in 10% dry milk diluted in Tris buffer saline ph 7.5 (TBS) with 0.1% Tween-20 (TBST). Membranes were incubated overnight at 4 C with mouse primary antibody against BMP-2 (5 μg/ml, R&D systems), BMP-4 (1:200, Santa Cruz), BMP-7 (4 μg/ml, R&D systems), β-actin (1:4000, Sigma) and rabbit primary antibody against psmad1/ 5/8 (1:500, Cell Signaling), Smad1/5/8 (1:1000, Santa Cruz), pstat3 (1:1000, Cell Signaling) and Stat3 (1:1000, Cell Signaling). All dilutions were made in TBST. Blots were rinsed in TBST three times for 10 min

3 Q. Xiao et al. / Experimental Neurology 221 (2010) each and incubated for 1 h at room temperature with secondary horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit IgG (1:1000, Cell Signaling). Peptides were detected on a high performance chemiluminescence film (GE Healthcare) by an Enhanced Chemiluminescence (ECL) Western blotting Kit (GE Healthcare). Images were digitally scanned and analyzed with Image J software (National Institutes of Health). Protein content was normalized according to β-actin content. Immunohistochemistry At 1, 2 and 4 days after injury, animals were killed by an overdose of pentobarbital sodium (100 mg/kg, i.p.), followed with transcardial perfusion of 0.9% saline for 5 min and then 4% paraformaldehyde (PFA, Sigma) in 0.1 M PBS (ph 7.4) for 15 min at room temperature. Two 3 mm-spinal cord segments, which were 1.5 mm rostral and 1.5 mm caudal to the lesion epicenter, were removed and postfixed in the same fixative for 2 h at 4 C before being cryopreserved in 30% sucrose solution in PBS for 2 3 days at 4 C. Spinal cords were frozen in OCT compound (Tissue Tek) and 15 μm transverse sections were cut and mounted on gelatin-coated glass slides. Sections were preincubated in blocking solution containing 8% normal donkey serum in the diluent solution (1% BSA and 0.3% Triton X-100 in 0.01 M PBS, ph 7.5) for 1 h at room temperature. Sections were then incubated overnight at 4 C with the following primary antibodies in the diluent solution (Table 1). Sections were then washed three times in 0.01 M PBS for 10 min each and then incubated with the following secondary antibodies (Table 2) diluted in 0.01 M PBS for 1 h at room temperature in the dark. Sections were also counterstained with the nuclear dye DAPI (1:10,000, Sigma) and mounted in fluorescence mounting medium (Dako). Negative controls consisted of secondary antibody alone. All samples were examined with a fluorescence microscope (Leica) and images were acquired with a digital camera (Diagnostic Instruments Inc.) and image capturing software (Diagnostic Instruments Inc.). For bromodeoxyuridine (BrdU) immunohistochemistry, mice received intraperitoneal injection of the thymidine analog BrdU (100 mg/kg; Sigma) solution at 1 day and at 2 h before sacrifice. Cryostat sections of the T9 spinal cord segments were processed to detect BrdU. Briefly, sections were pretreated in proteinase K (5 μg/ml) diluted in 0.01 M PBS for 10 min at room temperature, followed by 30 min in 2 M HCl at 37 C and 15 min in 0.1 M Borax buffer (ph 8.5) at room temperature. Nonspecific binding was blocked using 8% normal donkey serum in the diluent solution as described above. Sections were incubated overnight with mouse or rat anti-brdu Table 1 Primary antibodies used for immunohistochemistry. Name Dilution ratio Manufacturer Mouse anti-bmp-2 1:200 R&D Systems Goat anti-bmp-2 1:200 Santa Cruz Mouse anti-bmp-4 1:200 Santa Cruz Goat anti-bmp-4 1:200 Santa Cruz Mouse anti-bmp-7 1:200 R&D Systems Goat anti-bmp-7 1:200 Santa Cruz Rabbit anti-psmad1/5/8 1:400 Cell Signaling Mouse anti-nestin 1:100 BD Biosciences Mouse anti-neun 1:100 Millipore Corporation Mouse anti-map2 1:100 Sigma Goat anti-chat 1:100 Millipore Corporation Goat anti-doublecortin 1:100 Santa Cruz Rabbit anti-olig2 1:200 Millipore Corporation Mouse anti-gst-π 1:200 BD Biosciences Mouse anti-ng2 1:200 Millipore Corporation Rabbit anti-ng2 1:200 Millipore Corporation Rabbit anti-iba-1 1:500 Wako Mouse anti-gfap 1:200 Millipore Corporation Rabbit anti-gfap 1:200 Sigma Table 2 Secondary antibodies used for immunohistochemistry. Name Dilution Ratio Manufacturer Donkey anti-mouse Alexa Fluor 488 1:1000 Invitrogen Goat anti-mouse Alexa Fluor 568 1:1000 Invitrogen Donkey anti-rabbit Alexa Fluor 488 1:1000 Invitrogen Goat anti-rabbit Alexa Fluor 568 1:1000 Invitrogen Donkey anti-goat Alexa Fluor 488 1:1000 Invitrogen Donkey anti-goat Alexa Fluor 568 1:1000 Invitrogen antibodies (1:500, Roche, and 1:500, AbD Serotec, respectively) at 4 C, and washed three times in 0.01 M PBS for 10 min at room temperature. After primary antibody incubation, secondary donkey anti-mouse Alexa Fluor 488-conjugated (1:1000, Invitrogen) or donkey anti-rat Alexa Fluor 488-conjugated antibodies (1:1000, Invitrogen) were applied on the sections for 1 h at room temperature. Sections were counterstained with the nuclear dye DAPI (1:10,000, Sigma) and mounted in fluorescence mounting medium (Dako). The total number of proliferating cells per 15 μm section in the injured spinal cord was determined from the average number of BrdUpositive cells in coronal sections taken from 1.5 mm rostral and 1.5 mm caudal to the lesion epicenter. Representative sections were chosen from one of every 10 sections in accordance with a rostral-tocaudal direction. The proportion of proliferating cells per section was derived from the ratio of BrdU-positive cell numbers to DAPI-labeled nucleus numbers. The proportion of a specific population of proliferating cells (NSCs, astrocytes, OPCs and microglia/macrophage) was calculated by taking the number of specific cells doublelabeled with BrdU and dividing the number by the total number of BrdU-positive cells. Neurosphere culture To establish neurosphere cultures, spinal cords were isolated from 4- to 6-week-old mice. Thoracic spinal cords at T8 T10 were dissected in DMEM/F12 (1:1) with L-glutamine and 15 mm HEPES (Invitrogen) and cut into small pieces. Spinal cord tissue was dissociated in 0.05% trypsin (with EDTA. 4Na, Invitrogen) in Hanks' Balanced Salt Solution for 5 min in a 37 C water bath. The enzymatic reaction was stopped by the addition of 10% fetal bovine serum (FBS, Invitrogen), followed by centrifugation at 250 g for 5 min at room temperature. The pellet was re-suspended in serum-free medium (SFM) containing 20 ng/ml EGF (Sigma) and 5 ng/ml bfgf (Sigma), and triturated with a pipette to obtain a single-cell suspension. The cell suspension was cultured in a25cm 2 tissue culture flask (IWAKI) in SFM at 37 C with 95% air and 5% CO 2. The medium was changed every 3 days and neurospheres were passaged after 12 days in vitro. Neurospheres were dissociated by trypsin, and cells were subjected to the neurosphere culture again to form secondary neurospheres (P1) and obtain sufficient cell numbers for subsequent NSC differentiation analysis. To determine the effect of BMP on the differentiation of NSCs, P1 neurospheres were assessed for the presence of cell type-specific markers for neurons, astrocytes and oligodendrocytes. Nine days after culturing, P1 neurospheres were retrypsinized and triturated into single-cell suspensions. Single cells were plated in 4-well culture plates at a density of cells per coverslip; coverslips were coated with poly-l-ornithine (1:10 diluted in H 2 O, 350 μl/coverslip, Sigma) and laminin (5 μg/ml, Sigma). Cells were grown in the differentiation medium (1% FBS was added to the SFM without EGF or FGF) with or without the addition of BMP-4 (10 ng/ml or 30 ng/ml, R&D Systems) and cultured for 3 and 7 days under neural differentiation conditions, and quantitative analysis of the percentage of cells expressing neuronal and glial cell markers was performed. The medium was changed every third day in vitro. To test the effects of Noggin on NSC differentiation, neurosphere cultures were established from both normal uninjured and injured

4 356 Q. Xiao et al. / Experimental Neurology 221 (2010) spinal cords. The culture medium and culture period were the same as above. For neural differentiation, single cells from P1 neurospheres were separated into three groups: two groups were grown in the differentiation medium as described previously, with or without the addition of BMP-4 (30 ng/ml, R&D Systems), and the third group was pretreated with Noggin (200 ng/ml, R&D Systems) for 2 h before application of BMP-4 (30 ng/ml, R&D Systems) into the differentiation medium. All three groups were cultured for 3 days. After differentiation, cells were fixed with 4% PFA and processed for immunofluorescent staining. Cells were immunostained using the following antibodies in diluent solution overnight at 4 C: mouse anti- GFAP (1:1000, Millipore Corporation), mouse anti-β-tubulin III (1:500, Sigma), mouse anti-gst-π (1:500, BD Biosciences), and mouse anti- Nestin (1:500, BD Biosciences). After rinsing three times with 0.01 M PBS for 10 min, cells were incubated for 1 h at room temperature in the dark with secondary donkey anti-mouse IgG Alexa Fluor 488- conjugated antibody (1:1000, Invitrogen). Nuclei were counterstained with DAPI (Sigma). Coverslips were mounted on glass slides and examined under a fluorescence microscope (Leica). The number of positive stained cells was counted in the whole coverslip (three coverslips per medium condition) under the microscope. The proportion of a specific population of differentiated cells (astrocytes, oligodendrocytes and neurons) was calculated by dividing the number of specifically immunolabeled cells by the total number of DAPI + nuclei. In the Noggin-treated experiment, the ratio of GFAP + astrocyte to β-tubulin III + neuron numbers was calculated by dividing the proportion of astrocytes by the proportion of neurons. Statistical analysis Differences among multiple groups were analyzed by one-way analysis of variance (ANOVA) using Tukey's post hoc test for multiple group comparisons from SigmaStat software. A p value b0.05 was considered statistically significant. Error bars were used on graphs to represent the standard error of the mean (SEM). Results Enhanced expression of BMP-2/4/7 and psmad1/5/8 after spinal cord injury BMP-2, BMP-4, and BMP-7 mrnas (Figs. 1A, B) and protein (Figs. 1C, D) were detected in both the normal uninjured mouse spinal cord and injured spinal cords at 12 h, and 1, 2, 4, and 8 days after contusive injury. Densitometric analysis of the PCR products (Fig. 1A) showed that BMP-2, BMP-4 and BMP-7 mrna expression levels were significantly increased from 1 to 2 days, 12 h to 2 days, and 1 day after injury, respectively (Fig. 1B). Similar to the results of QT-PCR, Western blots analysis also demonstrated that expression levels of BMP-2, BMP-4, and BMP-7 proteins were all significantly increased at 1 and 2 days after injury compared with the normal uninjured control (Figs. 1C, D). Western blot analysis of samples of normal uninjured and injured mouse spinal cords showed increased psmad1/5/8 expression levels from 12 h to 4 days after spinal cord injury compared with normal uninjured controls (Figs. 1E, F). BMP-2/4/7 and psmad1/5/8 cellular localization in the injured spinal cord Immunohistochemistry showed the expression patterns of BMP-2, BMP-4, BMP-7 and psmad1/5/8 in the injured mouse spinal cord in relation to specific neuronal and glial markers. BMP-2, BMP-4 and BMP-7 immunoreactivity was observed in a variety of cell types in the spinal cord at 1 or 2 days after injury, when the BMPs mrna and protein expression levels were highest. In the gray matter, BMPs were mainly detected in Nestin + NSCs (Fig. 2A), MAP2 + mature neurons (Fig. 2B) and ChAT + motor neurons (Fig. 2C). In the white matter, BMPs were co-expressed with Olig2 in oligodendrocytes (Fig. 2D), and Iba-1 in microglia/macrophage (Fig. 2E). However, GFAPexpressing astrocytes did not demonstrate any BMP-2, BMP-4 or BMP-7 expressions (data not shown). Double-labeling immunocytochemistry identified potential target cells that might respond to BMPs after spinal cord injury. Expression of psmad1/5/8 was co-localized with cell-specific markers associated with NSCs and neural cells in the injured spinal cord. psmads were widely detected in NeuN + mature neurons (Fig. 3A), ChAT + motor neurons (Fig. 3B) and doublecortin + (DCX) immature neurons (Fig. 3C) in the gray matter. In the white matter, psmad immunostaining was localized in Nestin + NSCs (Fig. 3D), GFAP + astrocytes (Fig. 3E), NG2 + OPCs (Fig. 3F) and GST-π + mature oligodendrocytes (Fig. 3G). Possible sources of increased GFAP + cells after spinal cord injury are Nestin + NSCs, NG2 + OPCs and proliferating GFAP + resident astrocytes Injured spinal cord sections stained for GFAP and BrdU indicated one of the origins of the increased numbers of reactive astrocytes present after spinal cord injury. It was found that reactive astrocytes came from the proliferation of GFAP + resident astrocytes (Figs. 4A C). Other potential sources of astrocytes in the spinal cord were demonstrated using double immunolabeling studies of GFAP with Nestin and NG2. GFAP expression was detected in Nestin + (Figs. 4D F) and NG2 + cells (Figs. 4G I), suggesting that NSCs and OPCs in the injured spinal cord could also be the sources of the increased numbers of GFAP-expressing cells after spinal cord injury. Since all three types of cells (astrocytes, NSCs and OPCs) expressed psmad (Fig. 3), proliferation of resident astrocytes and differentiation of NSCs and OPCs towards GFAP + cells could be mediated by BMP signaling. Effect of BMP-4 and Noggin on the differentiation of NSCs isolated from adult mouse spinal cord Characterization and self-renewal property of NSCs derived from adult spinal cord Our immunohistochemical studies indicated that BMP and psmad expressions were detected in Nestin + cells after spinal cord injury. However, it was unclear whether these reactive Nestin + cells were NSCs. In further analyses of the effects of BMP-4 on cell differentiation in the spinal cord after injury, multipotent NSCs from the dissociated adult (4- to 6-week-old) normal uninjured and injured spinal cord were isolated and cultured in the presence of bfgf and EGF to establish neurospheres. NSCs formed clusters within 3 days of culture Fig. 1. BMP-2/4/7 and psmad1/5/8 expressions are significantly increased in the injured spinal cord. QT-PCR analyses showed enhanced BMP mrna expression levels in the injured spinal cord (A, B). A: Ethidium bromide-stained gels demonstrated QT-PCR products of BMP-2 (181 bp), BMP-4 (426 bp), BMP-7 (254 bp) and β-actin (227 bp). β-actin was used as an internal control for equal amounts of template cdna. B: Densitometric comparison of the average amount of cdna product of BMPs from normal uninjured and injured spinal cord samples. The data demonstrate that BMP-2 mrna was significantly upregulated at 1 and 2 days, BMP-4 mrna was notably increased at 12 h, and 1 and 2 days and BMP-7 mrna was markedly enhanced at 1 day after injury compared with normal uninjured control. Western blot analyses showed increased BMP protein expression levels in the injured spinal cord (C, D). C: Protein expression was visualized on a chemiluminescence film. The results indicated an upregulation of BMP-2/4/7 protein in the injured spinal cord. β-actin was used as an internal control for equal amounts of loaded protein. D: Densitometric comparison of BMP expression levels showed that all three types of BMP were significantly increased at 1 and 2 days after injury compared with normal uninjured control. Western blot analyses also showed increased psmad1/5/8 protein expression level in the injured spinal cord (E, F). E: Expression level of psmad1/5/8 detected on a chemiluminescence film was upregulated after injury. Smad1/5/8 and β-actin were used as internal controls for equal amounts of loaded protein. F: Densitometric comparison of psmads showed that protein expression level was significantly increased at 12 h, and 1, 2 and 4 days after injury compared with normal uninjured control. All data are mean±sem of three independent experiments (n=3). Significant difference in BMP mrna, BMP protein and psmad protein expression levels between injured and normal uninjured spinal cord (pb0.05).

5 Q. Xiao et al. / Experimental Neurology 221 (2010) and neurospheres were found at 8 9 days in vitro (DIV). Immunocytochemical staining showed that cells that contributed to neurosphere formation were immuno-positive for the NSC-specific marker Nestin (Fig. 5A). Dissociation of primary clones (P0) with trypsin into a single-cell suspension, followed by repetition of the neurosphere culture procedure, demonstrated the ability of these sphere-forming cells to self-renew and generate secondary clones (P1). Cells from the secondary to the eighth generation of sub-clone were all Nestinpositive, indicating that the progeny cells were NSCs (data not shown). In addition, cells from P1 neurospheres generated neurons

6 358 Q. Xiao et al. / Experimental Neurology 221 (2010) Fig. 2. BMP-2/4/7 were co-expressed with Nestin (A), MAP2 (B), ChAT (C) in the gray matter, and Olig2 (D) and Iba-1 (E) in the white matter of the spinal cord 1 day post injury (dpi). Expression of BMP-2/4/7 was shown with red immunofluorescence labeling using Alexa Fluor 568 in the cytoplasm (arrows). Expression of the NSC marker Nestin, mature neuron marker MAP2, and motor neuron marker ChAT were shown with green immunofluorescence labeling using Alexa Fluor 488 in the cytoplasm (arrows). Expressions of the oligodendrocyte marker Olig2 (nucleus, arrow) and microglia/macrophage marker Iba-1 (cytoplasm, arrow) were shown with green immunofluorescence labeling using Alexa Fluor 488. Nuclei were counterstained with DAPI (blue). In merged images, the yellow color represents co-localization of BMP-2/4/7 with specific cell markers (arrows). Boxes in the pictures show magnified merged images. Scale bar=30 μm.

7 Q. Xiao et al. / Experimental Neurology 221 (2010) Fig. 3. psmad1/5/8 was co-expressed with NeuN (A), ChAT (B) and DCX (C) in the gray matter, and Nestin (D), GFAP (E), NG2 (F) and GST-π (G) in the white matter of the spinal cord 4 days post injury (dpi). Expression of psmad was presented with red Alexa Fluor 568 immunolabeling (arrows) in nucleus. Expression of the mature neuron marker NeuN (nucleus, arrow), motor neuron marker ChAT and immature neuron marker DCX (cytoplasm, arrows) was shown with green immunofluorescence labeling using Alexa Fluor 488, in the gray matter. Expression of the NSC marker Nestin, OPC marker NG2, astrocytic marker GFAP and oligodendrocyte marker GST-π was shown with green immunofluorescence labeling using Alexa Fluor 488 in the cytoplasm and process (arrows), in the white matter. Nuclei were counterstained with DAPI (blue). In merged images, arrows indicate colocalization of NeuN, ChAT, DCX, Nestin, NG2, GFAP, and GST-π with psmad. Boxes in the pictures show magnified merged images. Scale bar=30 μm. (β-tubulin III + ), astrocytes (GFAP + ), and oligodendrocytes (GST-π + ) when induced to differentiate by culturing them on poly-l-ornithine/ laminin substrate (Figs. 5B J). These experiments demonstrated that cells isolated from the adult spinal cord have the capacity for selfrenewal and are multipotent, thus meeting the criteria for being NSCs. BMP-4 promoted the differentiation of astrocytes, while restricting the production of neurons and oligodendrocytes from NSCs in vitro Assessment of single cells from P1 neurospheres for the presence of cell type-specific markers for neurons, astrocytes and oligodendrocytes demonstrated the effect of BMP on the differentiation of NSCs. After 3 DIV, quantitative analysis of the percentage of cells expressing neuronal and glial cell markers showed that treatment with BMP-4 promoted astroglial differentiation. There was a significant increase in the proportion of cells expressing GFAP: 18.6±0.3% in the 30 ng/ml BMP-4 treated groups compared with the 11.7±0.6% in the absence of BMP-4 (pb0.05) (Fig. 5K). However, there was also a concomitant decrease in the percentage of cells differentiating toward an oligodendroglial or a neuronal lineage in the presence of BMP-4, when comparing the percentage of GST-π- and β-tubulin IIIexpressing cells grown in the absence of BMP-4. Compared with 1.72±0.06% GST-π + cells grown without BMP-4, there were only a total of 0.59±0.06% and 0.42±0.03% of the cells treated with 10 or 30 ng/ml of BMP-4, respectively, that expressed GST-π and exhibited an oligodendroglial morphology (pb0.05) (Fig. 5K). In addition, 4.29±0.57% and 2.19±0.57% of cells expressed β-tubulin III and displayed a neuronal phenotype with 10 or 30 ng/ml of BMP-4 treatment, compared with 7.58±0.32% of the cells grown in the absence of BMP-4 (pb0.05) (Fig. 5K). These results demonstrated that BMP-4 suppressed the differentiation of NSCs into oligodendrocytes and neurons while promoting astrocyte differentiation. Particularly, the morphology of astrocytes and oligodendrocytes cultured in the three types of medium used (1% FBS, 1% FBS+10 ng/ml BMP-4 and 1% FBS+30 ng/ml BMP-4) demonstrated the presence of both immature and mature cells. In all three types of medium, GFAP + astrocytes displayed two kinds of morphology: shorter, with fewer processes and longer, with more processes. In addition, unipolar, bipolar immature oligodendrocytes and multipolar mature oligodendrocytes were all detected in the three types of medium, demonstrating that BMP-4 had no effect on the maturation process of astrocytes and oligodendrocytes. However, neurons grown in medium containing BMP-4 had much shorter processes than those grown in medium without BMP-4 (Figs. 5D, G, J), suggesting that BMP-4 not only restricted neuronal differentiation, but also delayed neuronal maturation. After culture for 7 days, GFAP + astrocytes, GST-π + oligodendrocytes and β-tubulin III + neurons were still detected in all three types of cell culture. However, these cells exhibited more mature morphology when compared with the cells at 3 DIV. Cell counts showed that the proportion of astrocytes (25.5 ±0.88%) cultured in the

8 360 Q. Xiao et al. / Experimental Neurology 221 (2010) Fig. 4. Proliferation of GFAP-expressing cells in the spinal cord after injury. Immunofluorescence of BrdU is combined with labeling for the astrocytic marker GFAP (A C) 4 days post injury (dpi). A: Expression of GFAP was presented with red Alexa Fluor 568 immunolabeling in the cytoplasm (arrow). Nuclei were counterstained with DAPI (blue). B: Expression of the proliferation marker BrdU was shown with green immunofluorescence labeling using Alexa Fluor 488 in the nucleus (arrow). Nuclei were counterstained with DAPI (blue). C: In merged images, arrows indicate co-localization of BrdU and GFAP. In addition, GFAP was co-expressed with the NSC marker Nestin and OPC marker NG2 in the spinal cord at 4 dpi (D I). D, G: Expression of Nestin and NG2 was shown with red immunofluorescence labeling using Alexa Fluor 568. Nestin and NG2 were present in the cytoplasm (arrows). Nuclei were counterstained with DAPI (blue). E, H: Expression of GFAP was presented with green Alexa Fluor 488 immunolabeling, which was located in the cytoplasm (arrows). Nuclei were counterstained with DAPI (blue). F, I: In merged images, yellow color indicates co-localization of Nestin or NG2 with GFAP. Boxes in the pictures show magnified merged images. Scale bar=30 μm. medium containing 30 ng/ml BMP-4 was still the highest among the three types of medium, but there was almost no difference between the proportion of astrocytes cultured in the medium containing 10 ng/ml BMP-4 (18.5±0.88%) and that without BMP-4 (18.1± 0.48%). By contrast, the proportion of neurons cultured in the medium without BMP-4 (5.01±0.15%) increased significantly compared with those cultured in the media containing 10 ng/ml (1.43±0.03%) and 30 ng/ml BMP-4 (1.19±0.08%) (pb0.05). Furthermore, the proportion of oligodendrocytes cultured in the medium without BMP-4 was about 0.38%, and there were almost no oligodendrocytes detected when cultured in the BMP-4 containing media after 7 DIV (Fig. 5L). Noggin decreased the ratio of GFAP + astrocyte to β-tubulin III + neuron numbers after differentiation from NSCs in vitro P1 neurospheres from normal uninjured or injured spinal cord were dissociated and plated for neural differentiation. After 3 DIV, immunocytochemical and quantitative analysis showed that with Noggin there was a significant decrease in the ratio of the proportion of GFAP + astrocyte to the β-tubulin III + neuron numbers from normal (BMP-4 treated: 7.86±0.47; Noggin + BMP-4 treated: 3.78±0.56, p b0.05) and injured spinal cord (BMP-4 treated: 12.6±1.33; Noggin + BMP-4 treated: 2.95±0.30, pb0.05), suggesting that Noggin blocked the BMP-4 effect on promoting astrocyte production (Fig. 5M). The inhibitory effect of Noggin was more pronounced in neural cells derived from injured spinal cord than in those from normal uninjured spinal cord. Infusion of Noggin downregulated psmads expression, but did not decrease pstat3 and GFAP expression in the spinal cord after acute injury Our in vitro experiments have demonstrated that Noggin could block the effect of BMP-4 on promoting astrocyte differentiation from NSCs, we then studied whether blocking BMP signaling pathway by Noggin could perturb astrogenesis in the spinal cord after contusive injury. Western blot analysis showed that Noggin infusion by osmotic pump significantly decreased psmads expression at 4 days post injury (dpi), and this low expression lasted until 8 dpi, demonstrating that Noggin effectively attenuated the phosphorylation of Smad1/5/8. However, there was no significant change in GFAP expression level between Noggin- and vehicle-treated groups. Additional signaling pathways, e.g. JAK/STAT, could also regulate astrogenesis and compensate for the blocking of effect of BMP/Smad pathway on promoting GFAP expression. To examine the effect of Noggin on JAK/ STAT signaling pathway, we examined the expression level of pstat3, and found that Noggin treatment did not inhibit pstat3 expression (Fig. 6).

9 Q. Xiao et al. / Experimental Neurology 221 (2010) BrdU + proliferating cells increased after spinal cord injury The timing and cellular location of BrdU incorporation after spinal cord injury were demonstrated in animals that were injected with two doses of BrdU at 1 day and 2 h before they were killed. At 1 day after contusive injury, there was a significant increase of proliferating cells in the injured spinal cord compared with normal uninjured control (Fig. 7M), and the proportion of BrdU + cells reached its peak at 4 dpi (pb0.01). Co-labeling of BrdU and specific cell markers showed that BrdU + cells expressed the NSC marker Nestin (Figs. 7A C), the astrocyte marker GFAP (Figs. 7D F), the OPC marker NG2 (Figs. 7G I) and the microglia/macrophage marker Iba-1 (Figs. 7J L). Quantitation of BrdU-positive nuclei with several cell type-specific markers showed that the proportion of BrdU-incorporating NSCs and OPCs reached their peaks at 1 dpi and then began to decrease; BrdUpositive astrocytes significantly increased between 4 and 7 dpi. However, the number of proliferating microglia/macrophages remained at a high level until 7 dpi (Fig. 7N). Since NSCs, astrocytes, and OPCs all expressed psmad after injury (Fig. 3), it was possible that the increased expression of BMP-4 promoted cell proliferation in the spinal cord after injury. Discussion Injury induced upregulation of BMP-2/4/7 and psmad in the adult mouse spinal cord In the present study, QT-PCR and Western blot showed that BMP- 2, BMP-4 and BMP-7 mrna and protein expression levels were relatively low in the normal uninjured mouse spinal cord. However, both BMP mrna and protein expressions increased dramatically in the spinal cord after contusive injury. We also found that the expression level of psmad protein in the spinal cord was concomitantly increased after injury. Application of BMP-4 and BMP-7 has been shown to stimulate psmad1/5/8 expression at the demyelinating site (Fuller et al., 2007), and our data confirmed that psmad is the direct downstream signaling molecule in response to BMP stimulation. In addition, BMP-2, BMP-4 and BMP-7 expression levels began to decrease at 4 dpi and no significant difference was detected between the injured and normal uninjured spinal cord at 8 dpi, suggesting that expression of BMPs was downregulated at later stages of the spinal cord injury after the initial rise. Expression of psmad was also tightly correlated with the change of BMPs expression levels, returning to the normal level at 8 dpi. Significantly increased expression levels of BMPs and psmad, the two most important signaling molecules in the BMP pathway, suggested that BMPs might actively take part in the pathological or anatomical remodeling of the injured spinal cord, exerting pleiotropic effects on multiple cellular constituents of injured adult nervous tissues in a similar manner to the developmental events in the CNS. Phenotypic characterization of BMP-expressing and psmad-expressing cells in the adult mouse spinal cord after contusive injury In order to investigate the competency of BMPs in regulating cell reaction after injury, it is crucial to determine the cellular sources and targets of BMPs. Our study, for the first time, demonstrated the complete cellular localizations of BMPs and psmad in the injured adult spinal cord. BMP-2, -4 and -7 were expressed in Nestin + NSCs, MAP2 + mature neurons, and ChAT + motor neurons in the gray matter, and Olig2 + oligodendrocytes and Iba-1 + microglia/macrophages in the white matter. Previous work also revealed that BMP-7 mrna was located in motor neurons and oligodendrocytes (Setoguchi et al., 2001), and BMP-2/4 proteins were located in MOSP + oligodendrocytes, GFAP + astrocytes and CD11b + microglia/macrophages (Matsuura et al., 2008). We also found that psmad1/5/8 were localized in NeuN + mature neurons, doublecortin + (DCX) immature neurons and ChAT + motor neurons in the gray matter, and were extensively expressed in Nestin + NSCs, GFAP + astrocytes, NG2 + OPCs and GST-π + oligodendrocytes in the white matter. The fact that BMPs and downstream effector psmads were expressed in a variety of cell types indicated that BMP signaling pathway might be widely involved in controlling cell fate determination or regulating cellular components after CNS injury. Although Matsuura et al. (2008) reported BMP expression in GFAP + astrocytes, our examination of injured spinal cord did not reveal the presence of BMPs in GFAP + astrocytes. This could be due to the differences in animals used in the experiments (rat vs mouse), surgical modes, antibodies used or stages of injury. We observed that most neurons in the gray matter expressed psmad. Combined with results from previous studies which showed that apoptosis is usually induced in damaged neurons by deleterious factors after CNS injury (Nakahara et al., 1999), and that BMPs can protect neurons against neurotoxicity (Chou et al., 2008; Cox et al., 2004), we speculated that the activation of BMP pathways in neurons may participate in regulating the apoptosis process after injury. We also observed that psmad was expressed in Nestin + NSCs and GFAP + cells in the white matter (Fig. 3). In vitro experiments have demonstrated that BMPs mediated production and cellular maturation of astrocytes from adult mouse spinal cord-derived progenitor cells, while suppressing both neuronal and oligodendroglial lineage (Setoguchi et al., 2004). Combined with our observation that coexpressions of Nestin and GFAP, and BrdU and GFAP in reactive astrocytes occur in the spinal cord after injury (Fig. 4), these data implicated that BMPs not only directed NSCs to adopt an astrocytic fate but also promoted the proliferation of reactive astrocytes through psmad signaling in the injured spinal cord. In addition to the effect on astrocyte proliferation and differentiation, previous studies on cultured astrocytes from neonatal rat spinal cord demonstrated that BMP-4 and BMP-7 application increased CSPG protein expression (Fuller et al., 2007). These results, in addition to our findings, suggested that BMPs promote glial scar formation through several steps: 1) induction of NSCs to produce astroglial progenitors or astrocytes, 2) stimulation of astrocytic proliferation, and 3) upregulation of GFAP expression and CSPGs production in mature reactive astrocytes to form glial scar matrix. There were two other cell types expressing psmad in the white matter the NG2 + OPCs and GST-π + oligodendrocytes which were also possible targets of BMPs. BMPs have been shown to promote the production of astrocytes from OPCs at the expense of oligodendrocytes (Mabie et al., 1997). In addition, overexpression of Noggin, a BMP antagonist, decreases GFAP + cell production from OPCs (Kondo and Raff, 2004). Consistent with these results, we also found that a large number of NG2 + OPCs expressed GFAP after injury; this phenomenon was rarely observed in the normal uninjured spinal cord, supporting the notion that OPCs shifted to an astroglial lineage and provided an additional source of reactive astrocytes in the injured spinal cord. However, there was no significant decrease of oligodendrocytes in the spinal cord 1 week after injury. Our result of extensive co-labeling of BrdU and NG2 suggested that there was proliferation of NG2 + OPCs after injury. Therefore, it is possible that OPCs proliferated quickly after injury so that the proportion of oligodendrocytes did not decrease dramatically, though their lineage was inhibited to some extent. In addition, Mekki-Dauriac et al. (2002) also found that BMP-4 did not change the fate of already committed OPCs, indicating that only early-phase OPCs responded to BMPs. BMPs have also been shown to regulate psmad-expressing oligodendrocytes. In addition to the protective effect of BMPs on oligodendrocytes from apoptosis after spinal cord injury (Crowe et al., 1997), BMPs can prevent oligodendrocyte maturation. See et al. (2004) demonstrated that BMP-4 inhibits the expression of myelin proteins and myelin-associated glycoprotein in embryonic mouse

10 362 Q. Xiao et al. / Experimental Neurology 221 (2010) oligodendrocyte culture. Cheng et al. (2007) found that BMP-2 and BMP-4 can inhibit adult rat spinal cord-derived O1 + immature oligodendrocytes from developing into MBP + or CNPase + mature oligodendrocytes. This inhibitory effect may lead to the failure of axon remyelination after injury. In conclusion, BMPs can affect the development of the oligodendrocyte lineage after injury in two ways. First, BMPs inhibit OPC differentiation towards oligodendrocytes by blocking Olig1 and Olig2 in the Smad-Id-dependent pathway Fig. 5. BMP-4 promoted astrocyte differentiation, but restricted production of neurons and oligodendrocytes from adult spinal cord-derived NSCs. A: Primary neurospheres formed by the cells isolated from the adult mouse spinal cords after 12 days in culture. Expression of the NSC marker Nestin in the neurosphere cells was shown with green immunofluorescence labeling using Alexa Fluor 488. These NSCs from the secondary cloning were then assessed for the lineage-specific markers of neurons, astrocytes and oligodendrocytes (B J). After culture in the differentiating medium for 3 days, NSCs differentiated into three types of cell. B, E, H: GFAP + astrocytes were detected by green immunofluorescence labeling with Alexa Fluor 488 in 1% FBS medium in the absence of BMP-4 (B), with 10 ng/ml BMP-4 (E) and 30 ng/ml BMP-4 (H). C, F, I: GST-π + oligodendrocytes were detected by green immunofluorescence labeling with Alexa Fluor 488 in all three types of medium. D, G, J: β-tubulin III + neurons were detected by green immunofluorescence labeling with Alexa Fluor 488 in all three types of medium. Astrocytes (B, E, H) and oligodendrocytes (C, F, I) exhibited similar morphology in three types of culture medium, whereas processes of neurons grown in medium with BMP-4 were much shorter than those in medium without BMP-4 (D, G, J). K, L: After culture in the differentiation medium for 3 and 7 days, the numbers of three types of cells on the whole cover slip were counted. The relative proportion of GFAP + astrocytes grown in medium without BMP-4 was lower than that in medium with BMP-4 (10 or 30 ng/ml). However, proportions of GST-π + oligodendrocytes and β-tubulin III + neurons in medium without BMP-4 were higher than those in medium with BMP-4 (10 or 30 ng/ml). M: For NSCs from both normal uninjured and injured spinal cord, the ratios of GFAP + astrocytes to β-tubulin III + neurons were significantly lower in the medium pretreated with Noggin (200 ng/ml) 2 h before BMP-4 (30 ng/ml) than cells in the BMP-4 (30 ng/ml) treated medium after culture in vitro for 3 days. All data are mean±sem of three independent experiments (n=3). Significant difference in cell proportions between medium with and without BMP-4 (pb0.05).

11 Q. Xiao et al. / Experimental Neurology 221 (2010) Fig. 6. Western blot analyses showed that Noggin infusion by the osmotic pump distinctly reduced psmad1/5/8 protein expression level at 4 days after contusive injury in the spinal cord compared with vehicle diluent control; however, pstat3 and GFAP protein expression levels were not significantly changed during the same period in the injured spinal cord. Smad1/5/8, Stat3 and β-actin were used as internal controls for equal amounts of loaded protein. Fig. 5 (continued). (Samanta and Kessler, 2004). Second, BMPs prevent the maturation of oligodendrocytes by inhibiting the expression of myelin proteins. BMP-4 increased the production of astrocytes while suppressing the production of neurons and oligodendrocytes in the NSC culture prepared from adult mouse spinal cord Degeneration of neurons and failure of axon regrowth may be the two major reasons leading to functional loss after spinal cord injury. Adult spinal cord is not considered as a neurogenic region; even though quiescent NSCs in the spinal cord can be induced to differentiate into neural cells by injury, most of them only produce glial cells (Emsley et al., 2005). Because of this non-neurogenic microenvironment and the presence of deleterious factors such as BMPs, which inhibit neuron production after injury, multipotent NSCs transplanted into injured adult spinal cord usually differentiate into astrocytes instead of neurons. To address these issues, it is important to study the effects of BMPs on cell fate determination of NSCs in searching for effective reparative therapies for spinal cord injury. Most cultured NSCs have been isolated from embryonic or neonatal animals (Enzmann et. al., 2005; Liu et. al., 2004; Yanagisawa et. al., 2001); however, unlike embryonic or neonatal CNS cells, adult NSCs have different properties. Therefore, in our study, we chose adult spinal cord-derived NSCs to study the effect of BMP-4 since our in vivo experiments were performed on adult mouse spinal cord. We found that BMP-4 application enhanced the proportion of GFAP + astrocytes derived from cultured NSCs, whereas it prevented the production of GST-π + oligodendrocytes and β-tubulin III + neurons at 3 DIV and 7 DIV. Furthermore, a high dose of BMP-4 (30 ng/ml) had a more pronounced effect in promoting astrocyte production than a low dose (10 ng/ml). A great deal of previous work has reached a similar conclusion. Gross et al. (1996) found that BMP-2 induced the elaboration and differentiation of the astroglial lineage from SVZ progenitor cells, and the effect of BMP-2 on astrocyte differentiation was dose-dependent. Yanagisawa et al. (2001) observed that BMP-2, -4 and -7 have equivalent potential to increase astrocyte but decrease neuron number from cultured neuroepithelial cells. Together with our in vivo findings on the co-expression of psmad and the NSC-specific marker Nestin, we showed that the major effect of BMPs on NSCs after injury was probably to induce NSCs to differentiate into astrocytes. However, NSCs might respond to BMPs differently at different developmental stages. Li et al. (1998) found that neocortical precursors from the early embryonic period (E12) differentiated into MAP2 + neurons in response to BMP stimulation. Since BMPs may promote neuron differentiation at the early stage of embryonic development, we cannot rule out the possibility that BMP-4 promoted neuronal differentiation prior to 3 DIV. An alternative explanation is that BMP-4 only promoted astrocyte differentiation from adult spinal

12 364 Q. Xiao et al. / Experimental Neurology 221 (2010) cord-derived NSCs. Further work is needed to define the different roles of BMPs in neural cell specification. Furthermore, we observed that in BMP-4-treated group at 3 DIV, neurons displayed fewer and shorter processes, suggesting, for the first time, that BMP-4 may not only restrict neuronal differentiation, but also delay the maturation of neurons.