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Institut National de la Santé et de la Recherche Médicale (INSERM) U583, Physiopathologie et Thérapie des Déficits Sensoriels et Moteurs, Institut des Neurosciences de Montpellier, Hôpital Saint Eloi, Montpellier, France
Key Words. Neural stem cells • Spinal cord • Cytokines • Growth • Differentiation • Astrocytes • Gene array • Knockout
Correspondence: Jean-Philippe Hugnot, Ph.D., INSERM U583, INM-Hôpital Saint Eloi, 80 rue Augustin Fliche, 34295 Montpellier, France. Telephone: 00.33.4.99.63.60.08; Fax: 00.33.4.99.63.60.20; e-mail: hugnot{at}univ-montp2.fr
Received March 29, 2005;
accepted for publication September 6, 2005.
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ABSTRACT |
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, 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)–regulated 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.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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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 [11, 12]. Neural precursor cell fate is notably controlled by diffusible molecules among which neurotransmitters, retinoïds, and cytokines are the best characterized [13–15]. Cytokines represent a large family of small proteins that are secreted by a wide variety of cell types [16] and influence the behavior of target cells. With regard to neural precursor cells, cytokines of the interleukin-6 (IL-6) family and bone morphogenetic proteins (BMPs) have been shown to promote NS differentiation toward astrocytes [14], whereas platelet-derived growth factor (PDGF) and several neurotrophins positively influence the neuronal fate [17, 18]. Cytokines also control self-renewal of several types of stem cells, including hematopoietic and embryonic stem cells [19, 20].
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 [21]. 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.
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 [CNTF] 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.
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.
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MATERIALS AND METHODS |
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Spinal Cord NS Culture
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 [23]. Adult spinal cord NSs were derived from 3-month-old mice by using a protocol described in [24]. Full cell culture protocols are provided as supplemental online data.
X-Gal Staining
NSs derived from wild-type and knockin ENRB-lacZ embryos [22] 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°C in a 1 mg/ml X-Gal solution containing 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, and 2 mM MgCl2.
Bromodeoxyuridine Incorporation and Apoptosis Detection
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–mediated 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
Detailed protocols are provided as supplemental online data.
Fluorescence Quantification
Quantification of glial fibulary acidic protein (GFAP) content in astrocytes derived from wild-type and CNTF–/– 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–/– cultures were processed from three independent experiments.
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Gene-Array Analysis and RT-PCR
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.
Statistical Analysis
All results are expressed as means ± 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).
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RESULTS |
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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–D). 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– (supplemental online Fig. 1A). During CNS development, a large proportion of radial glial cells differentiate into astrocytes [26], 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).
Like forebrain NSs [28], 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.
NS Gene-Array Analysis
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).
Genes Expressed by Undifferentiated NSs
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 [LIFR]), CNTF (CNTFR), insulin-like growth factor (IGFs [IGFR1, IGFR2, and insulinR]), EGF (EGFR), FGFs (FGFR1–3), PDGFs (PDGFRa), neuregulins (erbB2/4), neurotrophins (TrkB/C), BMPs (BMPRI/II), and sonic hedgehog (Smoothened) [18]. More interestingly, we found receptors the expression of which has not yet been documented or fully established in neural precursor cells. These are receptors for the following cytokines/developmental factors: activins (Activin RI/II), apelin (APJ), stromal cell–derived factor-1 (SDF-1 [CXCR4]), endothelins (ENRB), glial cell line–derived neurotrophic factor (GDNF [GFRa1/2]), GM-CSF (GM-CSFRa), tumor necrosis factors (TNFs [TNF Rs]), gas6 (Mer, axl, dtk), macrophage-stimulating protein (MSPR), adrenomedullin and calcitonin gene–related peptide (CGRP [RDC-1]), 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 [29, 30]. NSs were found to express seven angiogenic factors: angiopoietin-1, ARP2, CTGF, netrin, PLGF, and VEGF-A and -B, with netrin displaying the strongest signal. This last developmental secreted factor was included in the angiogenic family because it was recently shown to have a strong angiogenic activity [31]. Chemokines are members of the cytokine family specifically involved in chemoattraction. As has long been known, chemokines regulate CNS leukocyte migration in pathological states, but more recent studies have shown that these proteins also have an important role in cellular communication in the developing and the healthy adult CNS [32]. 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 1–3, 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 2–5 (bind IGFs), LTBP1–3 (TGF-ß), Dan and chordin (BMPs), and Dkk-3 (wnts).
Genes Differentially Expressed after Differentiation
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 [33, 34], and thus their expression may be associated with these cells after differentiation. In regard to erbB receptors, we found that erbB2 expression did not vary on differentiation, whereas erbB4 showed a sharp increase (8.7-fold) and erbB3 was expressed only after differentiation. These results support those of Calaora et al. [35] and Kornblum et al. [36] showing that neural precursors of the ventricular/subventricular zones grown in NSs principally express erbB2/B4 receptors, whereas erbB3 appears to be confined to differentiated neurons and glial cells [37]. 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).
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Differential Influence of Exogenous Cytokines on NS Growth, Self-Renewal, and Differentiation
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 [39]). To confirm the expression of ENRB in CNS neural precursor cells, we derived NSs from embryonic spinal cord of a heterozygous mouse bearing an ENRB-lacZ knockin construct [22]. 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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, 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–treated 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– and IFN-–treated 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.
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.
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 [43], and it is possible that this peculiar mRNA may account for the observed discrepancy between the gene-array and Q-PCR techniques.
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). 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). 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.
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 [44] 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 [15, 45]. During CNS development, they participate in neuronal and glial differentiation [14] and exogenously added BMP can increase astrocyte differentiation in vitro and trigger smooth muscle cell differentiation of neural precursor cells [14, 46, 47]. These cytokines and related proteins such as TGF-ß are also known to be involved in the self-renewal of stem cells such as embryonic stem cells [48] and trophoblast stem cells [49]. To examine the putative role of endogenously detected BMPs in the NS model, NSs were grown or differentiated in the presence of the protein noggin. Noggin can bind to BMP-4, -6, -7, and -11 and behaves as an inhibitor of these proteins by modifying BMP-receptor interactions [45]. 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 [50]. In regard to neural precursor cells, exogenous CNTF has been shown to trigger strong astrocytic differentiation mediated by signal transducers and activators of transcription (STAT)-3 signaling [14]. In the presence of growth factors, CNTF is also implicated in the self-renewal of forebrain NSCs [51].
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–/– mice. These animals show a progressive loss of motoneurons [52] and display a poor recovery after experimental demyelination [53], and the basal expression of GFAP is reduced by 60% in the superior colliculus [40]. 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–/– 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–E). 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–/– 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–E). 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 [14]. However, even without exogenous CNTF, NSs readily differentiated into astrocytes, suggesting the existence of endogenous cytokines. We wondered whether endogenous CNTF, in addition to the endogenous BMPs (Fig. 1A), may take part in the NS differentiation process. First, we used double labeling to explore which cell types express CNTF after differentiation. As shown in Figure 1F, the majority of astrocytic GFAP+ cells stained strongly for CNTF. Fainter staining was also observed in neurons (ß-III-tubulin+) and oligodendrocytic cells (O4+) (data not shown). Interestingly, in a few cells, confocal imaging showed that CNTF was localized mainly in the nucleus (Fig. 1G) as reported in other models [54]. 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–/– 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–/–-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 (± 0.18, n = 3) in astrocytes derived from CNTF–/– NSs (Fig. 1I, right-hand histogram). To confirm this finding, GFAP expression was quantified by Western blot analysis. We found that upon differentiation, CNTF–/– 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–/– culture (n = 3, p < .05), in accordance with that measured by fluorescence image quantification. These results show that endogenous CNTF, in addition to the action of endogenous BMPs, has a role in regulating GFAP expression during NS differentiation.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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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 [19, 48]. Neural precursor cells, in addition to giving rise to neurons and/or glia for replacement purposes, appear to be beneficial to injured tissue by providing neurotrophic factors and/or by reducing glial scar [9, 57]. Some of the identified neurotrophic cytokines such as midkine [58], CNTF [50], and BMP-7 [59] and BMP inhibitors such as DAN are possible candidates for mediating these NS properties. Among detected, NS-expressed cytokines, angiogenic proteins are worth noting because recent studies have described the close interactions that exist between neural precursor cells and the vasculature. For instance, in the adult dentate gyrus, neurogenesis occurs close to vessels ("vascular niches") [30] and endothelial cells stimulate self-renewal and expand neurogenesis of NSCs [29]. Vascular endothelial growth factor (VEGF) is one of the cytokines that have been shown to mediate vascular-nervous system interactions, notably by enhancing neural precursor cell growth (reviewed in [60]). We report here that in the growing phase, NSs express seven genes for angiogenic factors (angiopoietin-1, ARP2, CTGF, netrin, placental growth factor (PLGF), and VEGF-A and -B). After differentiation, there was a considerable increase in transcription of PLGF and angiopoietin-1 (28- and 22-fold, respectively; Fig. 2A). The latter is a key protein involved in regulating vessel formation from endothelial cells and is expressed mainly by astrocytes during CNS development [61]. In addition to expressing angiogenic cytokines, NS cells expressed receptors for cytokines known to be expressed by endothelial cells—namely, neuropilin 1 and 2 coreceptors for VEGF, RDC-1, one of the putative receptors for the adrenomedullin—and receptor B for endothelins. Altogether, these results bring additional significant support for the existence of close relationships between neural precursors and endothelial cells.
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 [39]). Its expression in neural precursor cells was established using gene array, RT-PCR, and knockin mouse. Endothelins (ET-1, -2, and -3) are three small peptides originally purified from endothelial cells as potent vasoconstrictor peptides. Since then, they have been shown to be expressed by other cell types, including neurons, and their functions have been extended to the regulation of the proliferation and differentiation of several cell types. Notably, in the peripheral nervous system, ET-3 and receptor B play a critical role in regulating proliferation and differentiation of bi-potent melanoglial neural crest precursor cells [62]. In the CNS, endothelins stimulate astrocyte proliferation via receptor B activation. Because neural precursor cell growth is positively influenced by molecules secreted by endothelial cells [29], we selected endothelins as potential candidates for that function and showed that both ET-1 and -3 induced a 50% growth increase. This demonstrates that the endothelin system is present and able to be activated in cultured CNS neural precursor cells. Given that endothelins are expressed by endothelial cells in the CNS [39], our results suggest that, together with other identified factors such as VEGF, endothelins might take part in interactions between vascular and neural precursor cells.
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 [40] and BMP [41], the expression of which is typically increased in various CNS pathologies. To test for the influence of these cytokines, NS was grown in their presence with EGF/FGF2. A drastic reduction of growth (Fig. 3C) was observed, indicating that these receptors are functional in NS cells and that their activation can override the potent growth effect of FGF2/EGF. Similar results were reported using IFN-- and TNF- on newborn and adult rodent striatal cells grown in NSs [63, 64]. In agreement with our data, Gregg et al. [65] reported while this manuscript was in preparation that CNTF has a negative effect on self-renewal/expansion of embryonic spinal cord NSs and explants. In contrast, CNTF enhances growth of NSs derived from embryonic ventral forebrain [51, 65]. Thus, it appears that the response of neural precursor cells to a given cytokine depends on their original location and also on the stage of CNS development [66]. The CNTF-induced growth reduction is associated with a fourfold increase in the number of TUNEL+ cells in the culture, whereas the number of BrdU+ cells remains constant (Fig. 1C–E). This observed apoptosis is in accordance with data from Ben-Hur et al. and Monje et al. showing that other cytokines such as IFN-, IL-6, or activated microglia-conditioned media, induced cell death in rat striatal NS [63] and hippocampal precursor cell cultures [67]. 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-– and BMP-7–treated NSs. However, it is important to note that secondary NSs derived from BMP-7– or IFN-–treated 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 [OPCs] [68]). Assessment of the OPC number in NSs could help to determine how the tested cytokines acted. However, OPC markers such as A2B5 and NG2 are not specific to the oligodendrocytic lineage in NSs (our own unpublished data). IFN- and TNF- have been shown to block OPC proliferation and differentiation in vitro [69] or induce their apoptosis [70, 71], and thus similar effects could be responsible for the observed oligodendrocyte loss in NSs. On the other hand, the effects with BMP-7 and CNTF could be due to another mechanism, possibly cell conversion. In fact, our results are consistent with reports showing that BMPs, in vitro and in vivo, are able to strongly reduce oligodendrocyte formation [72, 73]. This occurs by decreasing the expression of the oligodendrogenic basic-helix-loop-helix (bHLH) transcription factor olig2 [74] and/or changing its intracellular localization and its sequestration by Id protein [75]. Recent results also indicated that CNTF, in combination with BMP-2, could decrease the generation of oligodendrocytes in vitro by reducing the level of olig2 [76]. Because in vivo loss of olig2 protein appears to convert OPC into astrocytes [77], it is plausible that a similar cellular conversion could account for the observed elevated astrocyte number in BMP-7–treated 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 [78, 79] 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.
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 [80], during zebrafish gastrulation [81], and in osteoblasts [82]. In regard to CNTF, FGF2 reduced CNTF mRNA level in astrocytic cultures [83]. 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 [84], proliferation, apoptosis [85], and differentiation [46]. Although noggin has been shown to bind to BMP-7 [45], we found no significant modification of NS growth and differentiation by addition of high concentrations of noggin during the growth phase. The role of this growth factor-regulated cytokine in NSs remains to be elucidated. Conversely, in the differentiation phase, adding noggin led to a sharp decrease of the number of astrocytic GFAP+ cells (Fig. 1A). This shows that in addition to the documented effect of exogenously added BMP-4, -6, and -7 on astrocytic differentiation [14, 86], 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.
CNTF is implicated in astrocytic differentiation and in induction of reactive gliosis [87]. In vitro, it induces astrocytic differentiation from oligodendrocyte progenitor cells and from other neural precursor cells [88, 89]. During NS differentiation, there is a considerable increase in CNTF protein expression (Fig. 4C), and because NS cells express CNTF receptor (supplemental online Table 1), we postulated that endogenous CNTF could play a major role in the generation of astrocytes from NSs. Using a null mouse for CNTF, we found that its contribution to NS differentiation was only moderate given that astrocytes, neurons, and oligodendrocytes were produced at a similar rate (Fig. 1H). However, GFAP protein expression as measured by Western blot and immunofluorescence was diminished (Fig. 1I, 1J). This suggests that, in NSs, endogenous CNTF is not responsible for specifying neural precursor cells to an astrocytic fate but could rather control astrocyte GFAP content. A similar conclusion was recently reached using different models [40, 90]. In a cortical slice assay, Morrow et al. showed that in E15 cortical precursor cells, glial fate specification is induced by FGF2 whereas CNTF only controls terminal glial differentiation [90]. Martin et al. reported that in CNTF null mice, the basal level of GFAP in the superior colliculus was reduced by 66% but that the absolute number of astrocytes was unchanged [40].
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 [63]) and by electron microscopy [91]. This cell death rate is likely to be influenced by endogenous CNTF given that NS cells null for this cytokine exhibit a reduced apoptotic rate that can be reversed by addition of exogenous CNTF (Fig. 1E). Cytokines have a well documented role in the control of precursor cell differentiation and growth, but their implication in regulating neural precursor cell death has been given little attention. However, the involvement of the IL-6 family of cytokines in apoptosis is well known in lactating mammary gland involution after weaning [92]. Actually, mice lacking LIF, IL-6, or STAT-3 (a key mediator of IL-6-family signaling) exhibit a delayed involution accompanied by a reduction of the cell death that normally occurs. Conversely, the implantation of LIF-secreting pellets into lactating mammary glands of wild-type mice increases apoptosis. In vitro, LIF can also induce apoptosis of several cell lines [92, 93]. Our data provide evidence for a role of endogenous and exogenous IL-6 family cytokines in regulating neuroepithelial apoptosis in vitro. The death of projecting neurons during nervous system development has been well described, but apoptosis also occurs in the neuroepithelium [94, 95], and several knockout mice for the apoptotic pathway (such as caspase 3 and 9 and apaf1) display pronounced early neural hyperplasia [94]. 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.
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.05). (J): Western blot analysis of GFAP expression in embryonic WT (WT lane) and CNTF–/– (KO lane) differentiated NSs. ß-Actin expression was used as an internal control. Quantification of GFAP/ß-actin ratio indicates a 2.5 ± 0.3 reduction in CNTF–/– differentiated NSs (n = 3, p < .05). Abbreviations: CNTF, ciliary neurotrophic factor; WT, wild-type; BrdU, bromodeoxyuridine; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling; GFAP, glial fibrillary acidic protein; PI, propidium iodide; KO, knockout; MW, molecular weight.
