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作者：Loc Deleyrolle, Sophie Marchal-Victorion, Ccile Dromard, Vanessa Fritz, Monique Saunier, Jean-Charles Sabourin, Christophe Tran Van Ba, Alain Privat, Jean-Philippe Hugnot作者单位：Institut National de la Sant et de la Recherche Mdicale (INSERM) U, Physiopathologie et Thrapie des Dficits Sensoriels et Moteurs, Institut des Neurosciences de Montpellier, Hpital Saint Eloi, Montpellier, France " o4 E# \1 g; C8 a
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      【摘要】
   Neurospheres (NSs) are clonal cellular aggregates composed of neural stem cells and progenitors. A comprehensive description of their proliferation and differentiation regulation is an essential prerequisite for their use in biotherapies. Cytokines are essential molecules regulating cell precursor fate. Using a gene-array strategy, we conducted a descriptive and functional analysis of endogenous cytokines and receptors expressed by spinal cord¨Cderived NSs during their growth or their differentiation into neuronal and glial cells. NSs were found to express approximately 100 receptor subunits and cytokine/secreted developmental factors. Several angiogenic factors and receptors that could mediate neural precursor cell¨Cendothelial cell relationships were detected. Among them, receptor B for endothelins was highly expressed, and endothelins were found to increase NS growth. In contrast, NSs express receptors for ciliary neurotrophic factor (CNTF), bone morphogenetic protein (BMP), interferon (IFN)-, or tumor necrosis factor (TNF)-, which, when added in the growth phase, led to a dramatic growth reduction followed by a reduction or a loss of oligodendrocyte formation on differentiation. In addition, NSs synthesize fibroblast growth factor 2/epidermal growth factor (FGF2/EGF)¨Cregulated endogenous cytokines that participate in their growth and differentiation. Notably, BMP-7 and CNTF were expressed during expansion, but upon differentiation there was a remarkable switch from BMP-7 to BMP-4 and -6 and a sharp increase of CNTF. Reintroduction of growth factors reverses the BMP expression profile, indicating growth factor-BMP cross-regulations. The role of endogenous CNTF was investigated by deriving NSs from CNTF knockout mice. These NSs have an increased growth rate associated with reduction of apoptosis and generate astrocytes with a reduced glial fibulary acidic protein (GFAP) content. These results demonstrate the combined role of endogenous and exogenous cytokines in neural precursor cell growth and differentiation.
      【关键词】 Neural stem cells Spinal cord Cytokines Growth Differentiation Astrocytes Gene array Knockout
   INTRODUCTION

The isolation of neural stem cells (NSCs) from the embryonic and adult central nervous system (CNS) of mammals is one of the major discoveries in the past decade in neurosciences. These cells are endowed with three cardinal properties: self-renewal, unlimited or extensive proliferation, and the ability to generate neuronal and glial cells (reviewed in .. ^5 k7 D' N0 ?" \. B" {4 b

To fully use their potential for repair and to direct their differentiation toward an appropriate cell fate, it is essential to better understand the cellular composition of NSs and to identify the molecules that regulate their growth, migration, and differentiation and the trophic factors they might secrete .
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Given the major role of cytokines in regulating precursor cell fate in various tissues, we anticipated that NSs might express several cytokine receptors that may influence their growth and differentiation on ligand activation. In particular, activation of these receptors by cytokines released during CNS lesion may influence the fate of resident or transplanted neural precursor cells . In addition, considering that NSs spontaneously differentiate in vitro into neurons and glial cells without any addition of exogenous factors, but simply by removing growth factors and providing adhesive substrate, we hypothesized that NSs might express growth-regulated endogenous cytokines that regulate their own differentiation. Such endogenous cytokines could possibly be responsible for the low rate of neurons produced by NSs on differentiation. Consequently, we first explored the variety of cytokines and their receptors which are expressed by NSs in their growth and differentiation phases, using gene-array screening and reverse transcription-polymerase chain reaction (RT-PCR). We used NSs derived from mouse embryonic and adult spinal cord, for which very little cellular and molecular characterization has been reported. To gain insight into the function of some identified endogenous cytokines and receptors, we then performed functional analyses by examining the effect of cytokines, cytokine inhibitors, and cytokine gene mutation on NS growth and differentiation.! X/ `0 r1 Z) q; {5 q: Q
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Our analysis demonstrates that NS cells express several receptors for pro-inflammatory cytokines and that their activation led to a severe decrease of NS growth and differentiation, in particular oligodendrocyte generation. NSs are also equipped with receptors and endogenous cytokines that could participate in their relationships with the vascular system. Notably, NSs express receptor B for endothelins (ENRB), and endothelins were found to stimulate NS growth. Finally, NSs express endogenous cytokines (ciliary neurotrophic factor  and BMPs), the level of which is radically modified between the growing and differentiating phases of NSs. Their expression is regulated by NSC growth factors (EGF and FGF2), and they participate in the growth and differentiation of NSs by regulating apoptosis and astrocytic differentiation.3 x$ }. |: S8 l: M0 \

These results illustrate that a combination of endogenous and exogenous cytokines regulate growth, death, and differentiation of neural precursor cells in vitro. As such, they provide several new insights on neural precursor cells which could help in designing better therapies for CNS pathologies.; Z$ k0 S. j5 ~5 a5 A
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MATERIALS AND METHODS
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Animals, _3 \7 F+ U, k% w
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Wild-type (C57BL6/J; Charles River Laboratories, Lyon, France, http://www.criver.com/crfrance), CNTF¨C/¨C (C57BL6/J; BRL/RCC, F¨¹llinsdorf, Switzerland, http://www.rcc.ch), and knockin ENRB-lacZ  mice were maintained at a temperature of 22¡ãC on a 12/12 hours light/dark cycle with free access to food and water.
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Spinal Cord NS Culture; x, M$ C1 I, R8 _2 u3 T8 {
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Embryonic spinal cord NSs free from dorsal root ganglions were obtained from embryonic day 13.5 (E13.5) mouse embryos (E0.5 = postcoitum) as described in . Full cell culture protocols are provided as supplemental online data.
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X-Gal Staining5 `: o- Q4 V( J( I5 D. M% D9 e

NSs derived from wild-type and knockin ENRB-lacZ embryos  were seeded at a density of 5 cells per µl. After 7 days of growth, NSs were fixed for 10 minutes with 0.2% glutaraldehyde, permeabilized with Triton 0.1% for 10 minutes, and stained for 12 hours at 37¡ãC in a 1 mg/ml X-Gal solution containing 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, and 2 mM MgCl2.) z2 D" `2 w+ r6 S% z

Bromodeoxyuridine Incorporation and Apoptosis Detection9 z) P' i7 @4 f6 q* q) r. d

To determine NS cell proliferation rate, 30 µM bromodeoxyuridine (BrdU) was added in the growth medium for 3 hours. NSs were then trypsindissociated, plated on polyornithine-coated coverslips by centrifugation, and immediately fixed for 15 minutes with 4% paraformaldehyde. For BrdU detection, coverslips were treated with 2N HCl for 30 minutes, washed with 0.1 M borate buffer, and incubated with rat immunoglobulin G (IgG) anti-BrdU (1:400; Abcam, Cambridge, U.K., http://www.abcam.com) and goat anti-rat conjugated to fluorescein isothiocyanante (FITC) (1:500; Jackson ImmunoResearch Laboratory, West Grove, PA, http://www.jacksonimmuno.com). Apoptosis was detected on non-HCl-treated coverslips by using the terminal deoxynucleotidyl transferase¨Cmediated dUTP-biotin nick-end labeling (TUNEL) method with the ApopTag fluorescein in situ apoptosis detection kit (Chemicon, Temecula, CA, http://www.chemicon.com) according to the manufacturer¡¯s instructions.

Immunofluorescence( s7 C7 L+ ]! S6 |/ x$ `/ u. F
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Detailed protocols are provided as supplemental online data.
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Fluorescence Quantification" ~# ]+ n1 A. u+ |
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Quantification of glial fibulary acidic protein (GFAP) content in astrocytes derived from wild-type and CNTF¨C/¨C differentiated NSs was performed using Scion Image Software (Scion Corporation, Frederick, MD, http://www.scioncorp.com). Photographs were computerized, and pixels with values higher than 55 (represented in red in Fig. 1I) were taken into account for quantification (pixel values range between 0 and 255, 0 being a black pixel). The GFAP intensity per astrocyte was then determined as the sum of all pixel values above background of all examined cells divided by the total number of examined cells. One hundred cells in control and CNTF¨C/¨C cultures were processed from three independent experiments.) {5 K/ z! g2 u
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Figure 1. Role of endogenous bone morphogenetic proteins (BMPs) and CNTF in neurosphere (NS) growth and differentiation. (A): NS differentiation in the presence of noggin. Recombinant noggin protein (100 ng/ml) was added during either the NS growing (7 days) or differentiation (4 days) phase. Differentiated cell quantification was carried out on day 4. Identical results were obtained when the differentiation process was extended to 6 days. Graph represents the percentage of immunoreactive cells for the indicated antibody found in the control (black bars) or noggin-treated (open and striped bars) culture. Values are the means ¡À SEM of three independent experiments. (B): Confocal micrographs of CNTF staining on undifferentiated spheres derived from WT (left) and CNTF¨C/¨C (right) embryos. Dotted white line indicates NS outline. Scale bars = 15 µm. (C): Cell number quantification of WT (black bars) and CNTF¨C/¨C (open bars) NSs grown in the presence of fibroblast growth factor 2/epidermal growth factor (FGF2/EGF) (20/20 ng/ml) with or without CNTF (10 ng/ml). Values are means ¡À SEM of three independent cultures. (D): Percentage of BrdU  cells in WT (black bars) and CNTF¨C/¨C (open bars) NSs grown with FGF2/EGF with or without CNTF. Values are means ¡À SEM of three independent cultures. (E): Percentage of TUNEL  cells in WT (black bars) and CNTF¨C/¨C (open bars) NSs grown with FGF2/EGF with or without CNTF. Values are means ¡À SEM of three independent cultures. (F): Expression of CNTF by astrocytes. Left- and right-hand images represent anti-GFAP and anti-CNTF staining, respectively. Nuclei are stained with Hoescht. Scale bars = 10 µm. (G): Images of one example of CNTF nuclear localization acquired by confocal imaging. The nucleus was counterstained with PI. Scale bar = 10 µm. (H): Differentiation of WT and CNTF¨C/¨C spinal cord embryonic NSs. Percentage of immunoreactive cells for the indicated antibodies after 4 days of differentiation is indicated for WT (black bar) and CNTF¨C/¨C (KO, open bar) NSs. Values are means ¡À SEM of three independent cultures. (I): Image quantification of astrocytic GFAP content in WT and CNTF¨C/¨C (KO) differentiated NSs. Images represent typical GFAP staining of WT and KO astrocytes (top) and corresponding pixels with values above background (bottom). Graph represents an estimation of GFAP content per cell deduced from image quantification. Values are GFAP intensity ¡À SEM. GFAP content per cells is reduced by 2.5 in CNTF¨C/¨C differentiated NSs. (A, C¨CE, I): Asterisks indicate statistical significance using an analysis of variance (ANOVA) test (p  .05). (J): Western blot analysis of GFAP expression in embryonic WT (WT lane) and CNTF¨C/¨C (KO lane) differentiated NSs. ß-Actin expression was used as an internal control. Quantification of GFAP/ß-actin ratio indicates a 2.5 ¡À 0.3 reduction in CNTF¨C/¨C differentiated NSs (n = 3, p 6 J6 m7 f8 j. ]/ S- E6 f
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Western Blot

Western blot tests were performed as described in detail in . Gels were loaded with 25¨C50 µg of proteins. Primary antibody dilutions were anti-CNTF (1:1000), anti-CNPase (mouse monoclonal IgG1, 1:1000; Chemicon), anti-GFAP (1:1000), anti-ß-III-tubulin (1:1000), and anti-ß-actin (mouse monoclonal IgG2a, 1:5000; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com).

Gene-Array Analysis and RT-PCR! n, b8 T7 @2 D' S" B, |5 ~; S

Detailed protocols are provided as supplemental online data.

Real-Time Quantitative PCR

Quantitative PCR (Q-PCR) was carried out using a light cycler (Roche Diagnostics, Mannheim, Germany, http://www.roche-applied-science.com) and a standard protocol with LightCycler FastStart DNA MasterPLUS SYBR Green I Kit (Roche Diagnostics). Primers were the same as those used for nonquantitative PCR; details of amplification conditions are available on request. Target gene expressions were normalized using ß-actin gene as an internal control. Each gene expression quantification was carried out on three independent experiments, and each sample was analyzed three times.
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Statistical Analysis

All results are expressed as means ¡À SEM. The statistical test used for comparisons was a one-factor analysis of variance (ANOVA) test with a 5% significance level. Statistical analysis was performed using Excel software (Microsoft Corporation, Redmond, WA, http://www.microsoft.com).2 x( Q; s% e/ j( H2 F

RESULTS6 M% G- w( n5 t7 _# f

Cellular Characterization of Embryonic Spinal Cord NSs
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NSs were derived from E13.5 mouse spinal cord in the presence of FGF2 and EGF. At this stage of development, the neural tube is closed and neural crest cells have emigrated. After a few days, spheres can be observed growing in suspension and can be dissociated and reseeded for more than 15 passages (data not shown). Because the cellular composition of embryonic spinal cord NSs has not been fully documented, we examined the expression of several cell-specific markers by immunofluorescence. The vast majority (> 90%) of cells expressed the immature neural marker nestin, and 76% of cells were found positive for RC2, a marker of radial glia , was detected in 60% of the cells (supplemental online Fig. 1A, 1C). The presence within the spheres of differentiated cells was explored by immunostaining for ß-III-tubulin (neuronal cells), O4 (oligodendrocytes), and GFAP-glutamine synthase-S100b (astrocytes and astroglia cells) (supplemental online Fig. 1A, 1B). We found a small percentage (! E! I/ h' }2 c* `

The spheres (hereafter referred to as "undifferentiated NSs") were then classically differentiated on an adhesive substrate for 4 days in the absence of growth factors. Nestin  and radial glia RC2  cells were hardly detectable after differentiation (0% and 2%, respectively). Neurons and oligodendrocytes detected by ß-III-tubulin and O4, respectively, accounted for 6% and 4% of the cells (supplemental online Fig. 1B¨CD). The majority of cells exhibited an astroglia phenotype as evidenced by high percentages of glutamine synthase  (92%), S100b  (53%), and GLAST  cells (75%). GLAST  cells were now RC2¨C (supplemental online Fig. 1A). During CNS development, a large proportion of radial glial cells differentiate into astrocytes , and considering their high percentage in undifferentiated NSs, more GFAP  cells would have been expected. We assumed that the cells expressing astroglial markers were differentiating radial glial cells that incompletely turned into astrocytes due to the limited amount of endogenous astrocytic-differentiation cytokines. We tested this possibility by adding CNTF or BMP-7 after 3 days of NS differentiation and assessing the number of differentiated cells on the fourth day. As shown in supplemental online Figure 1B, GFAP  cells now accounted for 90% of the cells whereas neuron number was not changed. Interestingly, the percentage of oligodendrocytes was significantly reduced by BMP-7.

NS differentiation was also confirmed by Western blots showing a sharp increase in astrocytic and oligodendrocytic marker expression (GFAP and CNPase, respectively) and a moderate increase in the ß-III-tubulin content (supplemental online Fig. 1E).8 e: x" m$ L1 W% {9 G2 b# G' f
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Like forebrain NSs , NSs derived from E13 mouse spinal cord are thus mainly composed of cells exhibiting a radial glial phenotype with a major tendency to differentiate in vitro into astroglial cells rather than into neurons or oligodendrocytes. These NSs predominantly contain undifferentiated cells that readily differentiate upon plating in absence of growth factors and are thus an appropriate model for carrying out detailed molecular characterization of spinal cord neural precursor cells in their growth and differentiation phases.+ m9 S8 e" k  p
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NS Gene-Array Analysis4 Q: p/ _0 D9 z' y) }3 h7 Y, ^
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To identify cytokines and cytokine receptors expressed by spinal cord precursor cells, we screened a gene array containing approximately 450 partial cDNAs coding for these proteins with probes derived from undifferentiated or differentiated NSs. The results of three independent experiments were analyzed, and 101 genes were found to be expressed in NSs (supplemental online Table 1). The amplitude of detected signals spanned two orders of magnitude, transforming growth factor (TGF)-ß-1 and TrkB expression being the lowest and highest, respectively. Analysis of gene expression by the array technique is prone to artifacts due to cross-hybridization with related members of the same gene family. To overcome this potential problem, we used RT-PCR to check the expression of genes scored positive in the array test, excluding those genes already described as being expressed in NS and neuroepithelial cells (references indicated in the footnote to supplemental online Table 1). Out of the 75 genes checked, 90% were confirmed positive by RT-PCR, thus demonstrating the specificity of the array analysis (supplemental online Table 1).
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Genes Expressed by Undifferentiated NSs2 p: p+ w: A) c

Receptors. Gene-array analysis showed the expression of receptors already known to be expressed in neural precursor cells. These include receptors for leukemia inhibitory factor (LIF ), Wnts (RYK), and Netrin (DCC). Several receptors for cytokines of the IL and interferon (IFN) families were also found to be expressed: receptors for type I (IFN- and -ß) and type II (IFN-) IFNs and receptors for IL-10 and IL-11.

Cytokines, Cytokine-Binding Proteins, and Secreted Developmental Proteins. Approximately 50 different genes coding for cytokines and secreted developmental factors were detected in undifferentiated NSs by gene-array analysis and confirmed by RT-PCR. The class of secreted factors that regulate angiogenesis is particularly interesting given that the existence of intimate relationships between neural precursor cells and endothelial cells has been recently described . Four chemokine genes were expressed by NSs: MIP3b, TECK, Fractalkine, and CRG-2. NSs were found to express several other cytokines in addition to angiogenic cytokines and chemokines: activinB, BMP-7 and -11, CNTF, m-CSF, FGFs, gas 6, Il-18, midkine, PDGF-, TGF-ß 1¨C3, TNF-, and THANK. Two members of the developmental glycoproteins Wnts (wnt 5a and wnt 5b) were strongly expressed. Finally, NSs also express several cytokine-binding proteins that can either facilitate or inhibit cytokine activity: IGFBP 2¨C5 (bind IGFs), LTBP1¨C3 (TGF-ß), Dan and chordin (BMPs), and Dkk-3 (wnts).
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Genes Differentially Expressed after Differentiation  g7 {/ |# q: F! r0 P6 [

After NS differentiation for 4 days, a more than twofold increase in expression was detected for 44 genes, whereas a decrease in expression was found for only seven genes (Fig. 2, black and grey bars, respectively). Twenty-seven and three variations, respectively, for the upregulated and downregulated genes were statistically significant. Of the genes that were differently expressed, only seven were uniquely expressed after differentiation: MCP-1, c-kit, OSM Rb, FGF-18, erbB3, and BMP-4 and -6. The chemokine macrophage-chemoattractant protein-1 (MCP-1) and the receptor for stem cell factor (c-kit) are expressed in neurons and glial cells under certain conditions . Finally, upregulation of BMP-4 and -6 after differentiation is particularly interesting because these molecules have been implicated in the differentiation of neural precursor cells (see below).

Figure 2. Differentially expressed genes. (A): Genes that are upregulated (black bars) after differentiation. (B): Genes that are downregulated (grey bars) after differentiation. Numbers within the bars indicate the ratio of differentiated to undifferentiated (A) signal and vice versa (B). If no signal could be detected in one of the two conditions, the ratio was considered superior to 100, and the corresponding bar is truncated. Asterisk indicates statistical significance using an analysis of variance (ANOVA) test (p  .05) between undifferentiated and differentiated signal values.

Among genes scored positive in undifferentiated NSs and which displayed a sharp increase in expression upon differentiation (> 10), two are implicated in angiogenesis: PLGF and angiopoietin-1. Another notable switch is that for FGFR2, the expression of which is upregulated more than 50-fold. Among the genes the expression of which was decreased upon differentiation, only three displayed a reduction that was statistically significant. Two of them encode IGF binding proteins (IGFBP2/3), indicating that these proteins are likely to be specific for immature proliferating cells. Even more striking was the 10-fold reduction in expression of BMP-7, a factor that appears to play many roles during development of the spinal cord .. Z! O4 X1 x) S4 B( s( n* N# v

Differential Influence of Exogenous Cytokines on NS Growth, Self-Renewal, and Differentiation
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One of the most highly expressed cytokine receptors detected by gene array in growing NSs is ENRB. Endothelins are cytokines notably expressed by endothelial cells that induce contraction of vascular muscles. However, their function extends beyond this role; endothelins have been shown to play active roles in regulating the migration, proliferation, and differentiation of neural crest precursor cells and astrocytes (reviewed in . After four passages, clonally grown NSs were stained with X-gal. Compared with wild-type NSs, approximately 50% of knockin NSs showed blue staining (Fig. 3A). Within spheres, staining was heterogeneous, suggesting that ENRB might be differently expressed by NS cells. To assess the effect of endothelin receptor on neural precursor cell growth, NSs were grown with FGF2/EGF and either endothelin-1 or -3. As shown in Figure 3B, a 1.5-fold increase in the number of cells was observed after 1 week. Use of endothelins alone, however, was not able to support NS growth (data not shown).
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Figure 3. Effect of exogenous cytokines on neurosphere (NS) growth, differentiation, and self-renewal. (A): Phase contrast micrographs of growing NSs derived from wild-type (left) and ENRB-lacZ (right) embryos. Spheres were stained with X-gal (dark staining). Scale bars = 10 µm. (B): Effect of endothelins on NS growth. Histograms display the cell number obtained from NSs grown for 1 week in the presence of fibroblast growth factor 2/epidermal growth factor (FGF2/EGF) alone or with ET-1 and -3 (50 ng/ml). Values are means ¡À SEM of three independent cultures. Asterisk indicates statistical significance using an analysis of variance (ANOVA) test (p  .05) between untreated and treated NSs. (C): Cell number quantification of NSs grown in the presence of FGF2/EGF alone or with the indicated proteins. Concentrations used were 50 ng/ml IFN- and TNF-, 10 ng/ml CNTF and BMP-7, and 100 ng/ml noggin. Values are means ¡À SEM of three independent cultures. Asterisk indicates statistical significance using an ANOVA test (p  .05) between untreated and treated NSs. (D): Differentiation of cytokine-treated NSs. Data shown are representative of three independent experiments. Indicated values are the ratio x 100 of the number of differentiated cells observed in treated NSs versus control NSs. Asterisk indicates statistical significance using an ANOVA test (p  .05) between untreated and treated NSs (n = 5). (E): Formation of secondary NSs from cytokine-treated NSs. Results shown are representative of three independent experiments. Indicated values are expressed in NSFU, which is the percentage of NSs formed per 100 cells seeded. Asterisk indicates statistical significance using an ANOVA test (p .05) between untreated and treated NSs (n = 7). Abbreviations: ENRB, endothelin receptor B; ET, endothelin; IFN, interferon; TNF, tumor necrosis factor; CNTF, ciliary neurotrophic factor; BMP, bone morphogenetic protein; EGF, epidermal growth factor; NS, neurosphere; NSFU, neurosphere-forming unit; GFAP, glial fibrillary acidic protein.3 R: t6 |; |" e# w/ @' c7 K$ R7 v: ^
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In addition to the endothelin receptor, this gene-array screening revealed the expression of several types of receptors for cytokines that are increased during acute or chronic CNS diseases: ILs, CNTF, BMPs, TNFs, and IFNs . To examine the functionality of these detected receptors and to explore how NS cells would respond to their activation, NSs were grown with FGF2/EGF for 7 days in the presence of BMP-7, CNTF, IFN-, and TNF-. As shown in Figure 3C, even in the presence of growth factors, addition of any of these cytokines led to a dramatic decrease of the neural precursor cell number. The most drastic effect was observed with BMP-7, leading to a 90% reduction. We next asked whether the treated NSs would give rise to equal amounts of all three neural lineages or whether these cytokines induce some fate restriction before differentiation is induced by growth factor removal. Treated NSs were thus differentiated, and the number of neurons, astrocytes, and oligodendrocytes was determined by immunofluorescence. As shown in Figure 3D, only BMP-7¨Ctreated spheres showed a small but significant increase in GFAP  cells. The production of neuronal cells was not significantly modified, although it tends to be lower for BMP-7 and higher for other cytokines. However, all cytokines diminished the number of oligodendrocytes, and remarkably, BMP-7¨C and IFN-¨Ctreated NSs did not generate these cells. Within NSs, only a small percentage of cells are able to form secondary spheres (between 0.1% and 1%, depending on the study). At least some of these cells are endowed with cardinal NSC properties, and secondary sphere formation is considered a good indicator of NSC self-renewing. We next evaluated whether cytokines would not only reduce NS growth but affect the number of these NS-initiating cells, by measuring the rate of formation of secondary spheres derived from cytokine-treated NSs. As shown in Figure 3E, no significant changes were observed with BMP-7, TNF-, and CNTF. We found that, in contrast, IFN- reduced the number of NS-initiating cells by almost half. The multipotency of the secondary spheres was checked by carrying out triple labeling on NSs differentiated at clonal density. Between 85% and 95% of tested NSs (15 per condition) were tripotent and gave rise to neurons, oligodendrocytes, and astrocytes. Together, these results indicate that exogenously added cytokines can positively or negatively influence growth and differentiation of embryonic spinal cord NSs.. {% A. N, M  F7 V8 K% P- @7 a

Role of Endogenous Cytokines CNTF/BMPs on NS Growth and Differentiation

The gene-array analysis showed the expression of several endogenous cytokines that are likely to participate in the neural precursor cell self-renewal, proliferation, and differentiation. Among them, CNTF and BMPs are candidates of interest because they have been reported to influence the fate of different types of stem cells. Whereas several studies have described the influence of these cytokines on NSs when added exogenously, almost nothing is known about the function of the corresponding endogenous cytokines. We therefore specifically examined the regulation of expression of endogenous cytokines BMP and CNTF and their contribution to NS growth and differentiation.
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Endogenous CNTF and BMP Expression Is Regulated by Growth Factors. A BMP switch operates during differentiation of NSs (supplemental online Table 1; Fig. 2). Undifferentiated NSs predominantly expressed BMP-7 and -11, whereas differentiated NSs turned off BMP-7, turned on BMP-4 and -6, and upregulated BMP-11. To confirm these results, we monitored expression of BMP-4, -6, and -7 by semiquantitative PCR and Q-PCR analysis (light cycler) using ß-actin as an internal control. Results in Figure 4A and 4B totally confirmed the gene-array analysis. BMP-4 and -6 were detected mainly in differentiated NSs, whereas BMP-7 was readily detected in the undifferentiated condition but barely expressed after differentiation. BMP-11 was expressed in both conditions but was slightly increased after differentiation (Fig. 4A). Q-PCR was also used to monitor CNTF mRNA expression during the differentiation process. As indicated in Figure 4B, CNTF mRNA was already expressed in growing NSs but showed a sixfold increase after differentiation. In contrast, the gene-array analysis indicated that the CNTF gene was expressed at the same level in both culture conditions (supplemental online Table 1). To gain further insight into this discrepancy, we analyzed CNTF gene expression at the protein level. In growing NSs, CNTF was detected in Western blots (Fig. 4C) but, in agreement with Q-PCR, there was a strong increase of protein level after 4 days of differentiation (Fig. 4C). In some tissues, a bicistronic messenger composed of the zinc finger protein Zfp91 and the CNTF sequences has been described , and it is possible that this peculiar mRNA may account for the observed discrepancy between the gene-array and Q-PCR techniques.5 }1 X) u, _1 m6 m; \, q2 _# ^& w3 o
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Figure 4. Endogenous CNTF and BMP expressions are regulated by growth factors. (A): Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of BMP and CNTF gene expression in undifferentiated neurospheres (NSs) (U), 4-day differentiated NSs (D), and differentiated NSs in which growth factors were reintroduced for 48 and 72 hours (D GF 48 and D GF 72). A unique band at the indicated size was obtained. ß-Actin amplification was used as control. (B): Quantification of BMP and CNTF mRNAs by quantitative polymerase chain reaction (Q-PCR) (light-cycler) of undifferentiated NSs, 4-day differentiated NSs, and differentiated NSs in which growth factors were reintroduced for 48 and 72 hours. Gene expressions were normalized with the ß-actin mRNA value. Values are means ¡À SEM of three independent cultures. For each culture, three Q-PCR quantifications were performed for each point. (C): Western blot analysis of CNTF expression in undifferentiated and differentiated NSs. NS CNTF is detected as a single band (approximately 23¨C25 kDa) comigrating with CNTF expressed in mouse sciatic nerve (4 µg). (D): Phase-contrast micrographs of undifferentiated NSs grown with FGF2/EGF (top left), 4 day-differentiated NSs (top right), and differentiated NSs in which growth factors were reintroduced for 48 and 72 hours (bottom images). (E): Formation of secondary NSs from control NSs or NSs formed after growth factor reintroduction (reformed NSs). Indicated values are expressed in NSFU ¡À SEM (n = 5), which is the percentage of NSs formed per 100 cells seeded. (F): Multipotency of secondary NSs derived from reformed NSs. Image shows the presence of oligodendrocytes (O4, blue), astrocytes (GFAP, green), and neurons (ß-III-tubulin, red) in a differentiated NS. (G): RT-PCR analysis of CNTF and BMP expression in undifferentiated and differentiated adult spinal cord NSs. Abbreviations: CNTF, ciliary neurotrophic factor; BMP, bone morphogenetic protein; GF, growth factor; FGF2/EGF, fibroblast growth factor 2/epidermal growth factor; NSFU, neurosphere-forming unit; GFAP, glial fibrillary acidic protein; MW, molecular weight.

The marked variation of CNTF and BMP transcripts upon differentiation suggested that the expression of these genes could be controlled by growth factors FGF2/EGF, which are removed during the differentiation assay. To explore this possibility, we reintroduced FGF2 and EGF in 4-day differentiated NSs and quantified CNTF and BMPs by Q-PCR at 48 and 72 hours. Interestingly, whereas after differentiation a single cell layer of differentiated cells was observed, reintroduction of FGF2/EGF in the culture caused a rapid reaggregation of cells and formation of spheres that detached from the dish and became free-floating (Fig. 4D). Gel analysis (Fig. 4A) and Q-PCR (Fig. 4B) indicated a reversal of CNTF and BMP mRNAs profiles back to that of undifferentiated NSs. Compared with differentiated NSs, FGF2/EGF triggers BMP-7 upregulation and downregulation of BMP-4, -6, and CNTF transcripts. We then asked whether these "reformed" NSs would give rise to new multipotent self-renewing NSs. Upon dissociation and clonal seeding, these generated secondary NSs at a rate equal to native NSs (Fig. 4E). These secondary NSs can also produce tertiary NSs (data not shown), indicative of their self-renewing, and upon differentiation and triple labeling, they generate neurons, astrocytes, and oligodendrocytes (14 out of 15 tested secondary NSs were multipotent) (Fig. 4F).+ f3 z; T- ]' \1 p% f9 n$ v1 W

These data strongly suggest the existence of cross-regulation between growth factors FGF2/EGF and BMP/CNTF cytokines in the NS model. In addition, the differentiation process appears to be reversible at the level of both endogenous cytokine profile and formation of new NSs.( F- A# I$ \5 G9 O
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Expression of CNTF and BMP in Adult Spinal Cord NSs. NSCs persist in several adult CNS regions called "niches." In rodents, adult spinal cord harbors NSCs located around the central canal  that can be grown using the classic NS assay. Adult-derived spinal cord NSs have phenotypic, growth, and differentiation properties very similar to E13.5 embryonic spinal cord NSs (our own data). To see whether the endogenous CNTF and BMP expressions and switches were characteristic of embryonic NSs or can also be observed in adult stem cells, we carried out semiquantitative PCR on growing and 4-day differentiated NSs derived from spinal cords of 6-month-old mice. As shown in Figure 4G, the expression profiles for CNTF, BMP-4, -6, and -7 were identical to those described in embryonic NSs. This demonstrates the conservation between embryonic and adult neural precursor cells of both expression and regulation of these endogenous cytokines.

Role of Endogenous BMP in Growth and Differentiation. BMPs are involved in the development of many organs and tissues, especially in the CNS . As shown previously, exogenous BMP-7 added in growth conditions drastically reduces NS growth (Fig. 3C). Considering the expression of endogenous BMP-7 transcript, we thus might have expected enhanced growth after noggin addition. However, the addition of noggin to the media, even at high concentration (100 ng/ml), did not lead to any detectable effect on NS growth (Fig. 3C). We next examined whether endogenous BMPs participate in NS differentiation. We added noggin, either during NS growth or during the differentiation process (4 or 6 days), and then immunofluorescence was carried out to determine the percentage of differentiated cells. No effect was detected using this BMP inhibitor during the growth phase (Fig. 1A), but a drastic effect was detected during the differentiating phase: there was a 90% decrease of astrocytic GFAP  cells, whereas the proportion of neuronal and oligodendroglial cell remained identical (Fig. 1A). Production of GFAP  astrocytic cells in NSs is thus likely to be regulated by endogenous BMPs expressed during the differentiation process.

Influence of Endogenous CNTF in NS Growth and Differentiation. CNTF was originally characterized as a survival factor for chick ciliary neurons in vitro .

Because both CNTF and the receptor for CNTF were expressed in NSs (supplemental online Table 1), we further explored the role of endogenous CNTF by deriving adult and embryonic spinal NSs from CNTF¨C/¨C mice. These animals show a progressive loss of motoneurons . After four passages, growth and differentiation of these NSs were compared with those of wild-type NSs. As shown in Figure 1B, CNTF expression was detected by immunofluorescence in growing NSs, whereas (as expected) no signal was found in mutant NSs. Influence of endogenous CNTF on NS growth was assessed by seeding dissociated embryonic mutant and control NSs at clonal density. As shown in Figure 1C, measurement of cell number after 7 days indicated that CNTF¨C/¨C culture yielded 70% more cells than controls. Similar results were obtained with wild-type and knockout adult-derived spinal cord NS (data not shown). The growth increase in mutant cultures could be reversed by adding exogenous CNTF (Fig. 1C), thus strongly suggesting that the lack of endogenous CNTF is responsible for the observed phenomenon. This difference of growth could be due to a modification of the cellular proliferation rate and/or cell death in culture. To address this issue, we measured the proliferation rate by BrdU incorporation and apoptosis by TUNEL assay in mutant and control cultures (Fig. 1D¨CE). Although we did not detect significant differences in the BrdU incorporation in mutant and control cultures (Fig. 1D), there was a fourfold reduction of TUNEL  cells in CNTF¨C/¨C cultures (4.8% and 1.3% for control and mutant cultures, respectively; Fig. 1E). This suggests that endogenous CNTF might induce apoptosis in NSs. This possibility was further evaluated by adding exogenous CNTF in growing wild-type and mutant NSs and measuring proliferation and apoptosis with BrdU and TUNEL assays (Fig. 1C¨CE). Addition of CNTF to cultures did not modify BrdU incorporation rate (Fig. 1D) but, in contrast, induced a striking increase in cellular apoptosis in both control and knockout cultures, reaching 21.5% and 9.15%, respectively (Fig. 1E). These data suggest that endogenous CNTF detected in adult and embryonic NSs is implicated in the control of neural precursor cell expansion by regulating apoptosis.

CNTF has been shown to be a strong inducer of neural precursor cell differentiation into astrocytes in vitro . Next, we examined the contribution of endogenous CNTF in the differentiation process by using immunofluorescence to determine the percentage of differentiated cells in wild-type and CNTF¨C/¨C embryonic NS culture. As shown in Figure 1H, the percentages of the different cell types were not significantly different in the two types of NS (same results were obtained with adult-derived spinal cord wild-type and knockout NSs; data not shown). However, whereas the number of astrocytic GFAP  cells was similar in the two cultures, we noted that CNTF¨C/¨C-derived astrocytes typically had a fainter GFAP staining (Fig. 1I, top pictures). This observation was confirmed by digital image quantification of GFAP fluorescence (Fig. 1I, bottom pictures). Using this method, we found that GFAP fluorescence was reduced 2.5 times (¡À 0.18, n = 3) in astrocytes derived from CNTF¨C/¨C NSs (Fig. 1I, right-hand histogram). To confirm this finding, GFAP expression was quantified by Western blot analysis. We found that upon differentiation, CNTF¨C/¨C NS culture expressed less GFAP than did wild-type NSs (Fig. 1J). With ß-actin as an internal control, gel quantification indicated a decrease of 2.5 in the GFAP/ß-actin ratio in CNTF¨C/¨C culture (n = 3, p / y* R' B! `0 J
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DISCUSSION

Adult- or embryo-derived NSs have been used extensively as a cellular source for transplantation in animals, and encouraging results have been obtained in animal models of spinal cord injury . These data call for a better understanding of endogenous and exogenous molecules that influence neural precursor cell differentiation and growth; such knowledge would ultimately be beneficial in designing more rational cellular therapies for CNS lesions. Thus, we considered it mandatory to better analyze neural precursor cells cultured in NSs¡ªin particular, the expression of cytokines and their receptors, because these proteins are key players in regulating many aspects of precursor cell fate, including proliferation, differentiation, migration, and apoptosis. Approximately 100 genes were found to be expressed, and the contribution of exogenous/endogenous cytokines on NS growth and differentiation was examined.( P0 h. W1 n( n. @* Y
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Three main conclusions can be derived from our analysis. First, we report here the expression by neural precursor cells of several endogenous cytokines, secreted developmental factors, and their receptors, the expression of which has not yet been documented in these cells. In several instances, both the cytokines and corresponding receptors (supplemental online Table 2) were found to be coexpressed, suggesting that autocrine and paracrine loops might be present in growing NSs. Some of them may participate in NSC self-renewal. In particular, BMP/BMPR gas6/Axl-Mer-Dtk loops might be implicated in NSC self-renewal given that these factors have been shown to mediate the same effect in embryonic stem and hematopoietic stem cells . In addition to expressing angiogenic cytokines, NS cells expressed receptors for cytokines known to be expressed by endothelial cells¡ªnamely, neuropilin 1 and 2 coreceptors for VEGF, RDC-1, one of the putative receptors for the adrenomedullin¡ªand receptor B for endothelins. Altogether, these results bring additional significant support for the existence of close relationships between neural precursors and endothelial cells.
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The second conclusion of this analysis is that, on activation, some of the identified receptors are able to enhance or reduce NS growth and differentiation, even in the presence of the growth factors FGF2/EGF. One remarkable receptor we detected is the G-protein-coupled ENRB (reviewed in , our results suggest that, together with other identified factors such as VEGF, endothelins might take part in interactions between vascular and neural precursor cells.3 F% ?: q0 w' R+ J- O' p

In addition to the endothelin receptor, gene-array screening allowed us to identify several cytokine receptors, notably receptors for proinflammatory cytokines such as TNF-, IFN-, and other cytokines such as CNTF . Thus, in addition to having a well documented role in neural precursor cell differentiation and proliferation, several cytokines are strong inducers of apoptosis.

Despite the severe reduction in cell number upon differentiation, only a slight variation or no variation was observed in the rate of astrocyte and neuron formation in cytokine-treated NSs compared with controls. Thus, it appears that the cytokine treatment has not radically changed the probability of the remaining cells adopting a neuronal or astrocytic fate during the differentiation process, or, alternatively, that the size of the putative astrocytic and neuronal progenitor populations in NSs have not been similarly reduced by the cytokines. In contrast, all cytokines strongly affected the number of oligodendrocytes, and no O4  cells were found in IFN-¨C and BMP-7¨Ctreated NSs. However, it is important to note that secondary NSs derived from BMP-7¨C or IFN-¨Ctreated NSs were able to generate oligodendrocytes upon differentiation, suggesting that the molecular modifications induced by the cytokines were not passed on to downstream NS generations. BMP-7 and CNTF could also reduce the oligodendrocyte production rate when applied 3 days after differentiation (supplemental online Fig. 1B). The tested cytokines could modify the number of oligodendrocytes by affecting either these cells or their precursors (i.e., the oligodendrocyte progenitor cells , it is plausible that a similar cellular conversion could account for the observed elevated astrocyte number in BMP-7¨Ctreated NS (Fig. 3D). Finally, it is worth noting that, in contrast to the oligodendrocyte reduction induced by exogenous cytokines, no effect was observed in NSs lacking endogenous CNTF (Fig. 1H) or cultured with a BMP inhibitor (Fig. 1A). This means that these endogenous cytokines, possibly due to their level or their accessibility, do not play a major role in oligodendrocyte formation in the NS model.

In addition to differentiation, we asked whether the cytokine treatment would affect the NS-initiating cells that represent only approximately 1% of NS culture. Their number was evaluated by measuring the percentage of secondary NSs arising from a given number of cells. This percentage gives the ratio of NS-initiating cells divided by total cells (i.e., NS-initiating cells plus non-NS-initiating cells), which can be simplified as NS-initiating cells divided by non-NS-initiating cells because the NS-initiating sphere number is small. This ratio was not significantly changed with BMP-7, CNTF, and TNF-. The fact that total cell number (Fig. 3C) decreased, whereas the ratio did not, indicates that cytokines reduce both cell types in NSs in equal or similar proportions. Interestingly, only treatment with IFN- showed a reduction of this ratio, suggesting that the NS-initiating cells might be preferentially affected by this cytokine. Because IFN- has been shown to affect hematopoietic and embryonic stem cells  by inducing their apoptosis or modulating their self-renewal, it is tempting to speculate that this cytokine could specifically affect NSCs by similar mechanisms. There are few data on the identity and properties of the NS-initiating cells at present, and further investigation into how cytokines affect their number would require the development of reliable markers. One of our remarkable findings is that, even after NS differentiation, cells able to form self-renewing multi-potent NSs can be rapidly retrieved by the addition of growth factors. This means that these NS-initiating cells either take no part in the differentiation process or de-differentiate in the presence of mitogens.4 A& q) @/ b  [. I& o# i9 F

The third conclusion we report here is the existence in adult and embryonic NSs of growth factor-regulated endogenous cytokines that are implicated in their growth and differentiation. In the growing phase, NSs expressed BMP-7, -11, and CNTF; then after differentiation, there was a sharp increase of CNTF, together with a reduction of BMP-7 and a concomitant drastic upregulation of BMP-4 and -6. Because NS differentiation is performed in the absence of growth factors, we hypothesized that the expression of these endogenous cytokines might be controlled by FGF2/EGF. Indeed, we found that reintroduction in the culture of FGF2/EGF led to a reversal of the endogenous cytokine profile. This suggests the existence of close cross-regulations between growth factors, CNTF, and BMP cytokines in neural precursor cells. Inhibition of BMP expression by FGF has been described in chick early epiblast . Thus, the existence of cross- talk between different families of cytokines is likely to be widespread and to play a major role in governing cell fate.

Because both BMPs and members of the IL-6 family have been shown to control stem cell proliferation and differentiation, we further explored the putative role of these cytokines. The high level of BMP-7 gene transcription in the growing phase suggests a role for this factor. BMP-7 is a pleiotropic cytokine that regulates cellular properties and fates such as expression of adhesion molecules , endogenous BMPs are main regulators of GFAP  cell formation during NS differentiation. This reduction in astrocytes was not counterbalanced by an increase of ß-III-tubulin  neurons. In addition, the presence of either a BMP inhibitor or exogenous BMP-7 during NS growth does not significantly modify the percentage of neurons produced (Figs. 1A, 3D). These data support the notion that the low level of neurons typically obtained from NS differentiation in vitro is unrelated to the presence of endogenous BMPs.6 L) F$ u/ w( y' p7 A8 l
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CNTF is implicated in astrocytic differentiation and in induction of reactive gliosis .: Y4 f8 n4 D) Y6 W" T
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We found that endogenous CNTF, in addition to participating in NS differentiation, negatively regulates NS expansion. We and others have observed that there was a consistent fraction of apoptotic cells in growing NSs, as identified by TUNEL staining (5% of apoptotic cells (Fig. 1E) and 15% in . There have been very few investigations of the initial mechanisms that induce this early neural cell death in vertebrates. The occurrence of spontaneous apoptosis in NS cultures and its modulation by endogenous and exogenous cytokine might be relevant in analyzing this phenomenon.. A. X  t8 P9 j. w
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DISCLOSURES

The authors indicate no potential conflicts of interest.

ACKNOWLEDGMENTS0 g9 C; _0 G6 q' k" b
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We are very grateful to Dr. N. Heintz (Rockefeller University, New York), who generously provided the BLBP antibody, Dr. P. Carroll (INSERM U583, Montpellier, France) for providing anti-CNTF antibody, and Drs. M.K. Shin (Fox Chase Cancer Center, Philadelphia) and Y. Kotolosev (University of Edinburgh, Edinburgh, Scotland) for providing knockin ENRB mice. We also thank Dr. Keith Langley (INSERM U583, Montpellier, France) for English corrections. This work was supported by Association pour la Recherche sur la Sclerose en Plaque (ARSEP), the Fundation Princesse Grâce de Monaco, the Institut pour la Recherche sur la Moelle Epini¨¨re (IRME), the Verticale Association, the Fondation pour la Recherche Medicale, E. Badinter, and the Association pour la Recherche sur le Cancer. S.M.-V., L.D., and C.D. are recipients of IRME, IRME/Demain Debout/ARSEP, and Demain Debout fellowships, respectively.
      【参考文献】6 D' H) ^9 G6 e3 a' R' r1 Z
; I. @2 }' s7 n7 C/ W

Gage FH. Mammalian neural stem cells. Science 2000;287:1433¨C1438.+ w0 D$ @8 H% i1 y

Alvarez-Buylla A, Garcia-Verdugo JM, Tramontin AD. A unified hypothesis on the lineage of neural stem cells. Nat Rev Neurosci 2001;2: 287¨C293.4 s/ E. w" n; d
3 R+ d& e' X; ^3 ~+ Y
Weiss S, Reynolds BA, Vescovi AL et al. Is there a neural stem cell in the mammalian forebrain? Trends Neurosci 1996;19:387¨C393.
+ i3 @/ y- m: Z) t! h" s
Seaberg RM, van der Kooy D. Stem and progenitor cells: The premature desertion of rigorous definitions. Trends Neurosci 2003;26:125¨C131.6 W7 ]4 s! T4 |1 E2 O. j! W
  k" }  [5 i7 c9 F" b+ X
Rao MS. Multipotent and restricted precursors in the central nervous system. Anat Rec 1999;257:137¨C148.) Z3 ^! N5 d' V

Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 1992;255:1707¨C1710.; x" Z* C* c# u) A7 R: t
$ h  n- b$ X6 w( Q4 A
Cao Q, Benton RL, Whittemore SR. Stem cell repair of central nervous system injury. J Neurosci Res 2002;68:501¨C510.
" m1 l0 R- b" y7 A
Lu P, Jones LL, Snyder EY et al. Neural stem cells constitutively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord injury. Exp Neurol 2003;181:115¨C129.' G8 J3 y) W" B) D2 E4 p

Pluchino S, Quattrini A, Brambilla E et al. Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. Nature 2003;422:688¨C694.
' |$ t* K8 e6 \- [
Ourednik J, Ourednik V, Lynch WP et al. Neural stem cells display an inherent mechanism for rescuing dysfunctional neurons. Nat Biotechnol 2002;20:1103¨C1110.

Gotz M. Glial cells generate neurons¨Cmaster control within CNS regions: Developmental perspectives on neural stem cells. Neuroscientist 2003;9:379¨C397.7 Q, |% t" x/ f: u5 W  M% F
$ s6 E: w  E+ Q4 Z/ k  R. `
Temple S. Stem cell plasticity¨Cbuilding the brain of our dreams. Nat Rev Neurosci 2001;2:513¨C520.

LoTurco JJ, Owens DF, Heath MJ et al. GABA and glutamate depolarize cortical progenitor cells and inhibit DNA synthesis. Neuron 1995;15:1287¨C1298.
+ b! O+ |0 S! K7 J9 R5 |
Nakashima K, Taga T. Mechanisms underlying cytokine-mediated cell-fate regulation in the nervous system. Mol Neurobiol 2002;25:233¨C244.
2 u3 ]. S$ z5 g2 N
Panchision DM, McKay RD. The control of neural stem cells by morphogenic signals. Curr Opin Genet Dev 2002;12:478¨C487.5 J' V  x3 q5 _$ I

Rothwell NJ, Luheshi G, Toulmond S. Cytokines and their receptors in the central nervous system: Physiology, pharmacology, and pathology. Pharmacol Ther 1996;69:85¨C95.- Y% Z) S" d3 T+ f' k

Caldwell MA, He X, Wilkie N et al. Growth factors regulate the survival and fate of cells derived from human neurospheres. Nat Biotechnol 2001;19:475¨C479.
5 G3 T+ R! B% d3 ?/ `
Benoit BO, Savarese T, Joly M et al. Neurotrophin channeling of neural progenitor cell differentiation. J Neurobiol 2001;46:265¨C280.7 `& Z% ^. s7 n9 G8 ?5 d
7 s: s- E  W8 a1 V9 h
Zhang J, Niu C, Ye L et al. Identification of the haematopoietic stem cell niche and control of the niche size. Nature 2003;425:836¨C841.7 A+ k; o: q) _! t. G, ^
; k3 j8 W# s2 {( v
Ying QL, Nichols J, Chambers I et al. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 2003;115:281¨C292.# H: v2 i3 @8 g0 w1 A7 M* z. c6 k0 C

Monje ML, Toda H, Palmer TD. Inflammatory blockade restores adult hippocampal neurogenesis. Science 2003;302:1760¨C1765.7 m  A$ H: V% \8 Z+ w9 p7 H+ J, q
$ t* h" _! V& N- e
Lee HO, Levorse JM, Shin MK. The endothelin receptor-B is required for the migration of neural crest-derived melanocyte and enteric neuron precursors. Dev Biol 2003;259:162¨C175.; @8 h. y: ^" V" b, j1 O9 d
% m" j0 Q' F* p
Yamamoto S, Nagao M, Sugimori M et al. Transcription factor expression and Notch-dependent regulation of neural progenitors in the adult rat spinal cord. J Neurosci 2001;21:9814¨C9823.

Johansson CB, Momma S, Clarke DL et al. Identification of a neural stem cell in the adult mammalian central nervous system. Cell 1999; 96:25¨C34.
1 K% ~/ t5 a0 B/ }- A
Marchal-Victorion S, Deleyrolle L, De Weille J et al. The human NTERA2 neural cell line generates neurons on growth under neural stem cell conditions and exhibits characteristics of radial glial cells. Mol Cell Neurosci 2003;24:198¨C213.+ ^, _" G5 ^/ x& ^* \
# V& c7 a! D; D8 i6 G
Malatesta P, Hack MA, Hartfuss E et al. Neuronal or glial progeny: Regional differences in radial glia fate. Neuron 2003;37:751¨C764.4 w. m# T; i& D  J$ G* A- F. w! C

Capela A, Temple S. LeX/ssea-1 is expressed by adult mouse CNS stem cells, identifying them as nonependymal. Neuron 2002;35:865¨C875.6 C8 f# M& p& Y1 p6 h

Hartfuss E, Galli R, Heins N et al. Characterization of CNS precursor subtypes and radial glia. Dev Biol 2001;229:15¨C30.! Y  L! t9 f, f
2 s$ K! i2 c: Z9 G: f+ j( {
Shen Q, Goderie SK, Jin L et al. Endothelial cells stimulate self-renewal and expand neurogenesis of neural stem cells. Science 2004;304:1338¨C1340.
5 d/ t. d) ~, U4 Q
Palmer TD, Willhoite AR, Gage FH. Vascular niche for adult hippocampal neurogenesis. J Comp Neurol 2000;425:479¨C494.

Park KW, Crouse D, Lee M et al. The axonal attractant Netrin-1 is an angiogenic factor. Proc Natl Acad Sci U S A 2004;101:16210¨C16215.

Asensio VC, Campbell IL. Chemokines in the CNS: Plurifunctional mediators in diverse states. Trends Neurosci 1999;22:504¨C512.$ N. z, p! N  v* A  d- `9 q
) [% d) q- w- l0 r0 W' N+ o
Zhang SC, Fedoroff S. Cellular localization of stem cell factor and c-kit receptor in the mouse nervous system. J Neurosci Res 1997;47:1¨C15.9 z3 |1 F, P/ C" e
4 ?% T( {" h4 n" K' I
Xia MQ, Hyman BT. Chemokines/chemokine receptors in the central nervous system and Alzheimer¡¯s disease. J Neurovirol 1999;5:32¨C41.9 ]2 D, @2 [. A3 g
, g, _3 |3 ^  z: M; [) b! m
Calaora V, Rogister B, Bismuth K et al. Neuregulin signaling regulates neural precursor growth and the generation of oligodendrocytes in vitro. J Neurosci 2001;21:4740¨C4751.3 J% @6 o: F( {& M7 t4 T3 ^8 E3 c
+ Q/ F2 t$ v0 o! E
Kornblum HI, Yanni DS, Easterday MC et al. Expression of the EGF receptor family members ErbB2, ErbB3, and ErbB4 in germinal zones of the developing brain and in neurosphere cultures containing CNS stem cells. Dev Neurosci 2000;22:16¨C24.
4 W# l( o/ o2 ~1 U; b& a6 i+ N
Gerecke KM, Wyss JM, Karavanova I et al. ErbB transmembrane tyrosine kinase receptors are differentially expressed throughout the adult rat central nervous system. J Comp Neurol 2001;433:86¨C100.; r- k3 ~+ s: y4 v

Butler SJ, Dodd J. A role for BMP heterodimers in roof plate-mediated repulsion of commissural axons. Neuron 2003;38:389¨C401.1 i- n* w/ l. i; w
4 l2 [+ C" t' M: J  |/ K
Mortensen LH. Endothelin and the central and peripheral nervous systems: A decade of endothelin research. Clin Exp Pharmacol Physiol 1999;26:980¨C984.
+ V5 E3 S/ K& W
Martin A, Hofmann HD, Kirsch M. Glial reactivity in ciliary neurotrophic factor-deficient mice after optic nerve lesion. J Neurosci 2003;23: 5416¨C5424.7 p& |' n' ^8 e9 W
$ I* r  }4 b4 n( H8 O+ @$ d
Setoguchi T, Yone K, Matsuoka E et al. Traumatic injury-induced BMP7 expression in the adult rat spinal cord. Brain Res 2001;921: 219¨C225.
5 C! {; P, N! _, z
Stoll G, Jander S, Schroeter M. Detrimental and beneficial effects of injury-induced inflammation and cytokine expression in the nervous system. Adv Exp Med Biol 2002;513:87¨C113.9 g. [! r3 \. P$ Z4 e

Saotome Y, Winter CG, Hirsh D. A widely expressed novel C2H2 zinc-finger protein with multiple consensus phosphorylation sites is conserved in mouse and man. Gene 1995;152:233¨C238.1 E, `4 M' X, x& G

Martens DJ, Seaberg RM, van der Kooy D. In vivo infusions of exogenous growth factors into the fourth ventricle of the adult mouse brain increase the proliferation of neural progenitors around the fourth ventricle and the central canal of the spinal cord. Eur J Neurosci 2002;16:1045¨C1057.

Balemans W, Van Hul W. Extracellular regulation of BMP signaling in vertebrates: A cocktail of modulators. Dev Biol 2002;250:231¨C250.- f8 ?. I% D% b1 I' `

Chang MY, Son H, Lee YS et al. Neurons and astrocytes secrete factors that cause stem cells to differentiate into neurons and astrocytes, respectively. Mol Cell Neurosci 2003;23:414¨C426.
. {/ }$ \! z+ z8 Q
Rajan P, Panchision DM, Newell LF et al. BMPs signal alternately through a SMAD or FRAP-STAT pathway to regulate fate choice in CNS stem cells. J Cell Biol 2003;161:911¨C921.
# h4 E0 H3 a* ~: x! W
Chambers I, Smith A. Self-renewal of teratocarcinoma and embryonic stem cells. Oncogene 2004;23:7150¨C7160.

Erlebacher A, Price KA, Glimcher LH. Maintenance of mouse trophoblast stem cell proliferation by TGF-beta/activin. Dev Biol 2004;275: 158¨C169.- {- }2 g! L& J! l1 l

Sleeman MW, Anderson KD, Lambert PD et al. The ciliary neurotrophic factor and its receptor, CNTFR alpha. Pharm Acta Helv 2000;74: 265¨C272.
7 c8 {5 q9 o; }: i
Shimazaki T, Shingo T, Weiss S. The ciliary neurotrophic factor/leukemia inhibitory factor/gp130 receptor complex operates in the maintenance of mammalian forebrain neural stem cells. J Neurosci 2001;21:7642¨C7653.

Masu Y, Wolf E, Holtmann B et al. Disruption of the CNTF gene results in motor neuron degeneration. Nature 1993;365:27¨C32.7 B. z4 `# d& W* T6 m0 o; T# h2 P
! D- Q1 T4 M5 {9 m$ n) B8 [' w
Linker RA, Maurer M, Gaupp S et al. CNTF is a major protective factor in demyelinating CNS disease: A neurotrophic cytokine as modulator in neuroinflammation. Nat Med 2002;8:620¨C624.

Bajetto A, Schettini G, Chimini G. Nuclear localization of ciliary neurotrophic factor in glial cells. Brain Res 1999;818:565¨C569.2 P4 M6 X2 r+ y$ }

Hofstetter CP, Holmstrom NA, Lilja JA et al. Allodynia limits the usefulness of intraspinal neural stem cell grafts; directed differentiation improves outcome. Nat Neurosci 2005;8:346¨C353.
( R7 Y0 r" j3 |; X# X+ M; M
Cao QL, Zhang YP, Howard RM et al. Pluripotent stem cells engrafted into the normal or lesioned adult rat spinal cord are restricted to a glial lineage. Exp Neurol 2001;167:48¨C58.1 g) `  c+ n0 Z% r

Llado J, Haenggeli C, Maragakis NJ et al. Neural stem cells protect against glutamate-induced excitotoxicity and promote survival of injured motor neurons through the secretion of neurotrophic factors. Mol Cell Neurosci 2004;27:322¨C331.

Yasuhara O, Muramatsu H, Kim SU et al. Midkine, a novel neurotrophic factor, is present in senile plaques of Alzheimer disease. Biochem Biophys Res Commun 1993;192:246¨C251.
* q: b& L# N% q7 K$ J
Yabe T, Samuels I, Schwartz JP. Bone morphogenetic proteins BMP-6 and BMP-7 have differential effects on survival and neurite outgrowth of cerebellar granule cell neurons. J Neurosci Res 2002;68:161¨C168.: q1 i( U, w! P  B- t4 l

Yasuhara T, Shingo T, Date I. The potential role of vascular endothelial growth factor in the central nervous system. Rev Neurosci 2004;15: 293¨C307.
# y! u* Z0 C. z
Acker T, Beck H, Plate KH. Cell type specific expression of vascular endothelial growth factor and angiopoietin-1 and -2 suggests an important role of astrocytes in cerebellar vascularization. Mech Dev 2001; 108:45¨C57.

Dupin E, Real C, Ledouarin N. The neural crest stem cells: Control of neural crest cell fate and plasticity by endothelin-3. An Acad Bras Cienc 2001;73:533¨C545.9 t2 {# ]4 j1 x! X! _& T
' s8 K: C2 Y: V& E+ j
Ben-Hur T, Ben-Menachem O, Furer V et al. Effects of proinflammatory cytokines on the growth, fate, and motility of multipotential neural precursor cells. Mol Cell Neurosci 2003;24:623¨C631.6 p9 ~4 I2 f! [3 r- `5 {
4 M# m+ H' E8 X9 p8 b* a
Wong G, Goldshmit Y, Turnley AM. Interferon-gamma but not TNF alpha promotes neuronal differentiation and neurite outgrowth of murine adult neural stem cells. Exp Neurol 2004;187:171¨C177.# j$ n# c) b, h
" A' m- }) n/ s( Z1 O7 o- u
Gregg C, Weiss S. CNTF/LIF/gp130 receptor complex signaling maintains a VZ precursor differentiation gradient in the developing ventral forebrain. Development 2005;132:565¨C578.- J. [4 ?5 i4 k% A  }$ i

Lillien L, Raphael H. BMP and FGF regulate the development of EGF-responsive neural progenitor cells. Development 2000;127:4993¨C5005.
+ e1 K9 M9 p8 O- b( F
Monje ML, Mizumatsu S, Fike JR et al. Irradiation induces neural precursor-cell dysfunction. Nat Med 2002;8:955¨C962.

Levine JM, Reynolds R, Fawcett JW. The oligodendrocyte precursor cell in health and disease. Trends Neurosci 2001;24:39¨C47.& X( z7 V" o, a8 `% U! r

Agresti C, D¡¯Urso D, Levi G. Reversible inhibitory effects of interfer-on-gamma and tumour necrosis factor-alpha on oligodendroglial lineage cell proliferation and differentiation in vitro. Eur J Neurosci 1996;8: 1106¨C1116.

Pang Y, Cai Z, Rhodes PG. Effect of tumor necrosis factor-alpha on developing optic nerve oligodendrocytes in culture. J Neurosci Res 2005;80:226¨C234.

Chew LJ, King WC, Kennedy A et al. Interferon-gamma inhibits cell cycle exit in differentiating oligodendrocyte progenitor cells. Glia 2005; 52:127¨C143.
/ M7 Q) \0 p  L# c% u" |5 T
Mabie PC, Mehler MF, Marmur R et al. Bone morphogenetic proteins induce astroglial differentiation of oligodendroglial-astroglial progenitor cells. J Neurosci 1997;17:4112¨C4120.
% N# L) G- r, [8 N$ j
Gomes WA, Mehler MF, Kessler JA. Transgenic overexpression of BMP4 increases astroglial and decreases oligodendroglial lineage commitment. Dev Biol 2003;255:164¨C177.% x. b, |7 V8 s. |+ H- x7 M

Mekki-Dauriac S, Agius E, Kan P et al. Bone morphogenetic proteins negatively control oligodendrocyte precursor specification in the chick spinal cord. Development 2002;129:5117¨C5130.) A8 C) P6 v) X
' y5 z( t! A6 I+ q& d
Samanta J, Kessler JA. Interactions between ID and OLIG proteins mediate the inhibitory effects of BMP4 on oligodendroglial differentiation. Development 2004;131:4131¨C4142.2 `% m( j  h$ z+ ^& N4 g' t2 F

Chojnacki A, Weiss S. Isolation of a novel platelet-derived growth factor-responsive precursor from the embryonic ventral forebrain. J Neurosci 2004;24:10888¨C10899.! a, Q  F4 u, V; E) E

Zhou Q, Wang S, Anderson DJ. Identification of a novel family of oligodendrocyte lineage-specific basic helix-loop-helix transcription factors. Neuron 2000;25:331¨C343.
& T" f# e5 m! S0 w- y
Zou GM, Reznikoff-Etievant MF, Hirsch F et al. IFN-gamma induces apoptosis in mouse embryonic stem cells, a putative mechanism of its embryotoxicity. Dev Growth Differ 2000;42:257¨C264.! J. Q& t' h! U6 p

Yang L, Dybedal I, Bryder D et al. IFN-gamma negatively modulates self-renewal of repopulating human hemopoietic stem cells. J Immunol 2005;174:752¨C757.
/ M* _4 _/ R$ H+ `- A4 H* Y
Wilson SI, Graziano E, Harland R et al. An early requirement for FGF signaling in the acquisition of neural cell fate in the chick embryo. Curr Biol 2000;10:421¨C429.

Furthauer M, Van Celst J, Thisse C et al. Fgf signaling controls the dorsoventral patterning of the zebrafish embryo. Development 2004; 131:2853¨C2864.

Pereira RC, Rydziel S, Canalis E. Bone morphogenetic protein-4 regulates its own expression in cultured osteoblasts. J Cell Physiol 2000; 182:239¨C246.% @1 f" u( w9 Z* t

Carroll P, Sendtner M, Meyer M et al. Rat ciliary neurotrophic factor (CNTF): Gene structure and regulation of mRNA levels in glial cell cultures. Glia 1993;9:176¨C187.: {' K" P! w7 p( h/ d6 i
; t/ n- F6 M' C, D* l/ d" T
Perides G, Safran RM, Rueger DC et al. Induction of the neural cell adhesion molecule and neuronal aggregation by osteogenic protein 1. Proc Natl Acad Sci U S A 1992;89:10326¨C10330.9 C0 W' \, q8 }! |. c

Baade Ro T, Utne Holt R, Brenne AT et al. Bone morphogenetic protein-5, -6 and -7 inhibit growth and induce apoptosis in human myeloma cells. Oncogene 2004;23:3024¨C3032.

Yanagisawa M, Nakashima K, Takizawa T et al. Signaling crosstalk underlying synergistic induction of astrocyte differentiation by BMPs and IL-6 family of cytokines. FEBS Lett 2001;489:139¨C143.$ }0 `$ x; ]" i0 \6 F
; \8 O& m1 c; F7 p3 ~/ }
Winter CG, Saotome Y, Levison SW et al. A role for ciliary neurotrophic factor as an inducer of reactive gliosis, the glial response to central nervous system injury. Proc Natl Acad Sci U S A 1995;92:5865¨C5869.! X2 p+ P' q; j! o

Hughes SM, Lillien LE, Raff MC et al. Ciliary neurotrophic factor induces type-2 astrocyte differentiation in culture. Nature 1988;335:70¨C73.
1 g9 w- h2 w  ^9 x2 G
Johe KK, Hazel TG, Muller T et al. Single factors direct the differentiation of stem cells from the fetal and adult central nervous system. Genes Dev 1996;10:3129¨C3140.
0 V& s* I0 O1 u: z# M
Morrow T, Song MR, Ghosh A. Sequential specification of neurons and glia by developmentally regulated extracellular factors. Development 2001;128:3585¨C3594.4 s9 @8 z0 \0 l& c1 N. n

Bez A, Corsini E, Curti D et al. Neurosphere and neurosphere-forming cells: Morphological and ultrastructural characterization. Brain Res 2003;993:18¨C29.  n) M! i. M9 J& j% [
$ d$ e6 }8 ]$ o. t4 \
Kritikou EA, Sharkey A, Abell K et al. A dual, non-redundant, role for LIF as a regulator of development and STAT3-mediated cell death in mammary gland. Development 2003;130:3459¨C3468.# {2 C# f' m4 H) C
* T+ N+ L0 v/ Y9 r% {5 q/ t* `
Minami M, Inoue M, Wei S et al. STAT3 activation is a critical step in gp130-mediated terminal differentiation and growth arrest of a myeloid cell line. Proc Natl Acad Sci U S A 1996;93:3963¨C3966.  u( o6 l! W8 L9 b1 s7 h

de la Rosa EJ, de Pablo F. Cell death in early neural development: Beyond the neurotrophic theory. Trends Neurosci 2000;23:454¨C458.& ^  G/ X  i9 ~  G) K

Hidalgo A, ffrench-Constant C. The control of cell number during central nervous system development in flies and mice. Mech Dev 2003;120:1311¨C1325.

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