Neurons

CNTF activates the JAK-STAT pathway in neurons in vitro (BUZAS et al., 1999).

Leptin regulates neuroproliferation both in vivo and in vitro and has neuroprotective effects through activation of JAK-STAT and thymoma viral proto-oncogene 1 (Akt) signaling (GARZA et al., 2008; TANG, 2008). SOCS2, 3 and 6 regulate neuronal differentiation and neurite outgrowth after induction of insulin-like growth factor-1 (IGF-1) and GH (GUPTA et al., 2011; TURNLEY et al., 2002). IL-9 signaling protects neonatal neurons form apoptosis by activation of the JAK-STAT pathway (FONTAINE et al., 2008). Jak2 and 3 mRNA are up-regulated in damaged hypoglossal motoneurons (YAO et al., 1997). STAT3 is massively expressed and activated in regenerating neurons after axonal injury (SCHWAIGER et al., 2000), and JAK1-STAT3 signaling is known to be involved in axonal pathfinding, branching, and muscle targeting in zebrafish motor neurons (CONWAY, 2006). Deletion of SOCS3, enables a robust and sustained axon regeneration (SMITH et al., 2009; SUN et al., 2011).

2.3.2 The JAK-STAT pathway in pathological conditions of the CNS 2.3.2.1 Inflammation

Tyrosine-protein kinase Lyn (Lyn), a member of the rous sarcoma oncogene (Src) family of protein tyrosine kinases is mainly expressed in hematopoietic cells (YAMANASHI et al., 1989), and neural tissues (UMEMORI et al., 1992). Lyn is associated with various cell surface receptor proteins including the B cell antigen receptor (BCR; YAMAMOTO et al., 1993) and CD40 (REN et al., 1994), triggers rapid signaling cascades such as the JAK-STAT pathway leading to cell proliferation and

differentiation (WANG et al., 2007). STAT1 signaling is observed in the early phase of B cells stimulation (SU et al., 1999). It is suggested that STAT5 plays a role in the self-renewal of B cells (SCHEEREN et al., 2005). BCR and Lyn-mediated activation of STAT3 is JAK-independent in B cells (WANG et al., 2007). IL-12 family (IL-12, IL-23 and IL-27) cytokines secreted by dendritic cells (DCs) play an immune-regulating role by the induction of Th1, Th2, Th17 or Treg cells (EGWUAGU C, 2013). The JAK-STAT signaling pathways are involved in the signaling of multiple of these pro- and anti-inflammatory cytokines. For instance, IL-12 activates JAK2-STAT4 signaling which promotes T cell differentiation into Th1 cells that secrete pro-inflammatory cytokines such as IFN-γ,   TNF-α and IL-6 (EGWUAGU C, 2013; ZHU et al., 2010).

Mice lacking STAT1 are susceptible to develop EAE, which suggests that STAT1 induction of Th1 and IFN-γ   are   essential   for   the   inflammatory   disease  in the CNS (BETTELLI et al., 2004; NISHIBORI et al., 2004). Moreover, pro-inflammatory cytokines including TNF-α,  IL-6, IL-17 and IL-22 are produced by Th17 cells, which is induced by IL-23-JAK2-STAT3 signaling (EGWUAGU C, 2013). Treg cells secrete anti-inflammatory cytokines such as IL-10, TGF-β1, and are activated by the IL-27-JAK1-STAT3 pathway (EGWUAGU C, 2013; REGIS et al., 2008). To some extent, expansion of Th1 and Th17 cells is involved in the pathogenesis of MS and EAE (AMADI-OBI et al., 2007; LAURENCE and O'SHEA, 2007). SOCS1 inhibits expansion of Th17 cells as observed in EAE (AHMED et al., 2010; FLOWERS et al., 2004). In brain ischemia IL-6 regulates survival-associated gene expression, via the JAK2-STAT1/STAT3 pathway (PLANAS et al., 2006).

2.3.2.2 Neoplasia

STAT3 signaling exhibits a dual role in glial tumorigenesis. STAT3 is considered a tumor suppressor due to its functional relationship to the major tumor suppressor phosphatase and Tensin homolog (PTEN). STAT3 is stimulated by phosphorylation of LIFR-β  and supresses IL8 expression, thereby inhibiting glioma cell proliferation and invasiveness (DE LA IGLESIA et al., 2008a). In contrast, Akt is constitutively activated in PTEN-deficient tumor cells, thus inhibiting FOXO3 function, and leading to LIFR-β   down-regulation, followed by lacking STAT3 activation and subsequent IL-8

up-regulation which drives glioma cell proliferation and invasiveness (DE LA IGLESIA et al., 2008a; DE LA IGLESIA et al., 2008b; DE LA IGLESIA et al., 2009). Furthermore, mutation of epidermal growth factor receptor class III variant (EGFRvIII) can lead to glioma formation (ZHANG et al., 2007). STAT3 and EGFRvIII regulate the transcription of oncogenic genes targets by the formation of a physical complex which can bind to oncogenic genes in nucleus (DE LA IGLESIA et al., 2009). Aberrant activation of STAT3 signaling is critically involved in glioblastoma pathogenesis, such as constitutive STAT3 activation in glioblastoma cells (IWAMARU et al., 2007;

RAHAMAN et al., 2002). Possible ligands of STAT3 signaling in glioblastomas include IL-6 and oncostatin M (VAN WAGONER and BENVENISTE, 1999). Inhibition of STAT3 signaling by JAK inhibitors AG490, WP1066 (IWAMARU et al., 2007;

RAHAMAN et al., 2002), and the STAT3 inhibitor PIAS3 (BRANTLEY and BENVENISTE, 2008) trigger apoptosis and inhibits survival of astroglioma cells in vitro (KONNIKOVA et al., 2003). Moreover, the IL-8-chemokine (C-X-C motif) receptor 2 (CXCR2)-STAT-3 signaling pathway is a regulatory mechanism promoting proliferation of astroglial tumor cells and regulating angiogenesis (KORKOLOPOULOU et al., 2012; PIPERI et al., 2011). Increased JAK1 expression was seen in low-grade gliomas (CATTANEO et al., 1998). STAT6 is also overexpressed in gliomas and promotes invasive growth (MERK et al., 2011).

2.3.2.3 Demyelinating diseases

A reduced severity of demyelination can be induced in EAE by blocking IL-12 signaling in T cells via the JAK-STAT pathway (MUTHIAN et al., 2006; NATARAJAN and BRIGHT, 2002). In MS, increased interferon-beta (IFN-β) induces STAT1 activation, which is considered a possible pathway that enhances the immune response (FENG et al., 2002). STAT1 proteins are markedly up-regulated in neurons and microglia in demyelinating EAE (JEE et al., 2001). The increased transcription of Stat1 RNA is widely distributed in neurons, astrocytes and microglia (MAIER et al., 2002). In MS patients activated STAT1 (FENG et al., 2002; FRISULLO et al., 2006;

GOBIN et al., 2001) mediates interferon signaling and is controlled by SHP-1 (FRISULLO et al., 2006; GOBIN et al., 2001; MASSA and WU, 1996; YETTER et al.,

1995). Lower levels of SHP-1 in macrophages of MS patients may result in activation of STAT1 and downstream target genes (CHRISTOPHI et al., 2009). Moreover, multiple studies suggest that the activation of the STAT1 signaling pathway by IFN-γ   signaling is involved in the pro-inflammatory changes in MS (DAVID et al., 1995;

FENG et al., 2002; MASSA et al., 2000). Up-regulation of activated STAT1, 3 and T-bet was found in peripheral blood CD4⁺ T-cells and monocytes in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and relapsing-remitting MS patients (MADIA et al., 2009). STAT3 enhances T cell differentiation and activity, which is a potential pathogenic factor in MS (LILL et al., 2012). Up-regualated STAT3 immunoreactivity in astrocytes is suggested to play an immune suppressive role in EAE (JEE et al., 2001). In vitro, increased expression of STAT3 signaling in SOCS3-deficient macrophages leads to shift these cells towards a M1 phenotype.

Adoptive transplantation of M2 macrophages into SOCS3-deficient mice with EAE ameliorates the disease process by an inhibition of STAT3 activation (QIN et al., 2012).

Massive expression of STAT4 and IL-12 in microglia is thought to accelerate the autoimmune inflammation in acute EAE (MATSUMOTO et al., 1992). TMEV-infection of Stat4 knockout mice results in delayed onset of severe demyelination after 180 days. In contrast, TMEV-infected Stat6 knockout mice show little demyelination at this time point (RODRIGUEZ et al., 2006). STAT6 activation and expression of STAT6-responsive elements in macrophages may play an important role in the pathophysiology of MS (CHRISTOPHI et al., 2009). Plumbagin inhibits EAE by affecting the differentiation of pathogenic Th1 and Th17 cells differentiation through a down-regulation of JAK-STAT signaling (JIA et al., 2011)

3. Highly malignant behavior of a murine oligodendrocyte precursor cell line following transplantation into the demyelinated and nondemyelinated central nervous system.

Hansmann F, Pringproa K, Ulrich R, Sun Y, Herder V, Kreutzer M, Baumgärtner W, Wewetzer K

Department of Pathology, University of Veterinary Medicine Hannover, Hannover, Germany.

Abstract:

Understanding the basic mechanisms that control CNS remyelination is of direct clinical relevance. Suitable model systems include the analysis of naturally occurring and genetically generated mouse mutants and the transplantation of oligodendrocyte precursor cells (OPCs) following experimental demyelination. However, aforementioned studies were exclusively carried out in rats and little is known about the in vivo behavior of transplanted murine OPCs. Therefore in the present study, we (i) established a model of ethidium bromide-induced demyelination of the caudal cerebellar peduncle (CCP) in the adult mouse and (ii) studied the distribution and marker expression of the murine OPC line BO-1 expressing the enhanced green fluorescent protein (eGFP) 10 and 17 days after stereotaxic implantation. Injection of ethidium bromide (0.025%) in the CCP resulted in a severe loss of myelin, marked astrogliosis, and mild to moderate axonal alterations. Transplanted cells formed an invasive and liquorogenic metastasizing tumor, classified as murine giant cell glioblastoma. Transplanted BO-1 cells displayed substantially reduced CNPase expression as compared to their in vitro phenotype, low levels of MBP and GFAP, prominent  upregulation  of  NG2,  PDGFRα,  nuclear  p53,  and  an  unaltered expression of signal transducer and activator of transcription (STAT) 3. Summarized environmental signaling in the brain stem was not sufficient to trigger oligodendrocytic differentiation of BO-1 cells and seemed to block CNPase expression. Moreover, the

lack of the remyelinating capacity was associated with tumor formation indicating that BO-1 cells may serve as a versatile experimental model to study tumorigenesis of glial tumors.

Cell Transplant. 2012; 21(6): 1161-75 doi: 10.3727/096368911X627444. Epub 2012 Mar 8

4. Signal transducer and activator of transcription 3 represents a molecular switch inducing astroglial instead of oligodendroglial differentiation of oligodendroglial progenitor  cells  in  Theiler’s  murine  encephalomyelitis

Yanyong Sun*^†, Arno Kalkuhl^‡, Ulrich Deschl^‡, Wenhui Sun*^†, Karl Rohn^§, Wolfgang Baumgärtner*^†, Reiner Ulrich*

*Department of Pathology, University of Veterinary Medicine Hannover, Germany,

†Centre for Systems Neuroscience Hannover, Germany, ^‡Department of Non-Clinical Drug Safety, Boehringer Ingelheim Pharma, Biberach (Riß), Germany, and

§Department of Biometry, Epidemiology and Information Processing, University of Veterinary Medicine Hannover, Germany

Manuscript in preparation

Running title: STAT3 in TME

Keywords: Janus kinase; Microarray; Multiple sclerosis; Oligodendrocyte progenitor cell; Signal transducer  and  activator  of  transcription;;  Theiler’s  murine  

encephalomyelitis

Correspondence: Dr. med. vet. Reiner Ulrich. Ph.D., Department of Pathology, University of Veterinary Medicine Hannover, Bünteweg 17, D-30559 Hannover, Germany. Tel: +49511-953-8670; Fax: +49511-953-8675; E-mail:

Reiner.Ulrich@tiho-hannover.de

Abstract

Aims: Insufficient oligodendroglial differentiation of oligodendroglial progenitor cells (OPCs) is responsible for remyelination failure and astroglial scar formation in Theiler's murine encephalomyelitis (TME). The aim of the present study is to identify molecular key regulators of OPC differentiation in TME, and to proof their mechanism of action in vitro. Methods: TME virus (TMEV) infected SJL/J-mice were evaluated by rotarod analysis, histopathology, immunohistology, and gene expression microarray analysis. The signal transducer and activator of transcription (STAT)3 pathway was activated using meteorin and inhibited using STAT3 inhibitor VII in the glial progenitor cell line BO-1 in vitro. Results: Immunohistology demonstrated a progressively decreasing percentage of myelin basic protein-positive white matter area in TME. In contrast, intralesional nerve/glial antigen 2 (NG2)-positive OPCs as well as glial fibrillary acidic protein (GFAP)-positive astrocytes were increased. Gene Set Enrichment Analysis revealed 26 Gene Ontology terms including Janus kinase (JAK)-STAT cascade to be significantly positively correlated with the density of NG2-positive OPCs. Immunohistology revealed an increased amount of activated, phosphorylated STAT3 (p-STAT3)-expressing cells within the lesions. Most p-STAT3-expressing cells co-expressed GFAP, followed by CD107b and NG2.

Activation or inhibition of STAT3-signaling in BO-1 cells cultured in astrocytic differentiation medium enhanced GFAP- or CNPase-expression, respectively.

Conclusions: Activation of the STAT3 pathway in OPCs is a key regulator shifting their differentiation from an oligodendroglial towards an astrocytic fate, thereby inducing astrogliosis and insufficient remyelination in TME.

Introduction

Multiple sclerosis (MS) is an etiologically unresolved, demyelinating central nervous system (CNS) disease, affecting more than 2.5 million patients worldwide [1]. MS is characterized by an infiltration of inflammatory cells with varying amounts of demyelination, remyelination, astrogliosis and axonal injury [1-3]. Pathomorphological changes in active demyelinating MS lesions suggest different pathomechanisms and a heterogeneous etiology of the disease [4]. Active demyelinating MS plaques can be divided in lesions suggestive of a primary immune-mediated pathogenesis and those possibly due to primary oligodendropathy. Accordingly, 30% of MS cases display an extensive destruction of oligodendrocytes, whereas in the remaining 70%, oligodendrocyte numbers are only reduced in areas of active myelin destruction and reappear in remyelinated areas [5]. A subset of approximately 20% of all MS patients exhibits extensive remyelination, whereas especially those patients with a progressive clinical course display only limited or absent remyelination [6-9]. The demyelinated axons in these chronic lesions are known to be vulnerable to secondary progressive axonal degeneration and atrophy [1,10,11]. Experimental studies show that the proliferation and differentiation of oligodendrocyte progenitor cells (OPCs) is a prerequisite for remyelination and functional recovery, whereas surviving oligodendrocytes are relatively quiescent and do not participate in remyelination [12,13]. Many MS lesions are known to contain OPCs. However, they frequently lack differentiation to myelinating oligodendrocytes [8,14-17]. A similar situation of a progressive demyelination with insufficient remyelination despite the presence of an at least transiently increased number of intralesional OPCs is reported in the Theiler´s murine encephalomyelitis (TME) virus (TMEV)-induced model of demyelination [18].

In contrast to TME, a fast and complete remyelination can be observed in most other models of MS, including experimental autoimmune encephalomyelitis (EAE) [19], cuprizone toxicity [20], lysolecithin-induced demyelination in the caudal cerebellar peduncle [21], and murine hepatitis virus (MHV) A-59 strain infection of mice [22], which renders TME to be the most useful model to investigate chronic progressive demyelination with remyelination failure. The principal reasons for a failure of

remyelination can either be a depletion of OPCs, inability of OPCs to proliferate and/or differentiate within the lesion due to a non-permissive environment; axonal loss or the inability of chronically demyelinated axons to be remyelinated [18,23,24].

Cumulative experimental evidence currently suggests that a block of oligodendroglial differentiation seems to represent the key factor in remyelination failure in MS [17,24].

Based on previous own experiments that suggested a shift from oligodendroglial towards astrocytic differentiation of OPCs in TME [18,25], we modified the hypothesis of TMEV-infection directly or indirectly induces a dysregulation of the differentiation of oligodendrocyte progenitor cells (OPCs) resulting in a shift from oligodendrocytic towards an astrocytic fate. Therefore the aims of present study were: 1.) to identify the principal molecular pathways regulating the differentiation of OPCs in TME employing gene expression microarrays, 2.) to verify the cellular source and activation status of the most promising candidate pathway in situ, and 3.) to demonstrate the suggested molecular switch-like mechanism of action in vitro.

Materials and Methods Laboratory animal procedures

Mice were housed in individually ventilated cage systems (Tecniplast, Hohenpeißenberg, Germany), under controlled conditions as previously described [18,26-28].

Five-week-old female SJL/JHanHsd-mice (Harlan Winkelmann, Borchen, Germany) or SJL/JCrl-mice (Charles River Laboratories, Sulzfeld, Germany) were inoculated into the right cerebral hemisphere with 1.6×10⁶ or 4.6x10⁷ plaque forming units per mouse of the BeAn-strain of TMEV or vehicle control (mock-infection), respectively, in two independent experiments, as previously described [18,26,27].

The clinical course was evaluated with a rotarod assay (RotaRod Treadmill, TSE Technical & Scientific Equipment, Bad Homburg, Germany), as previously described [27,29,30]. The rod speed was linearly increased from 5 revolutions per minute (rpm) towards 55 rpm over a time period of 5 min. All living mice were repeatedly assayed at 0 days post infection (dpi, pre-infection) and multiple time points post infection. A

mean score per mouse was calculated from three trials per time point. The attained rpm at drop was analysed for significant differences between TMEV- and mock-infected mice employing independent pair-wise Mann-Whitney U-tests (IBM SPSS Statistics, Version 20, IBM Corporation, Armonk, NY, USA). Statistical significance was designated as p 0.05.

Groups of six mice were necropsied at multiple time points up to 245 dpi as previously described [18,26,30]. The spinal cord was immediately dissected and representative cervical, thoracic and lumbar segments were fixed in 10% formalin, decalcified in ethylenediaminetetraacetic acid solution and embedded in paraffin wax. The remaining parts of the spinal cord were removed from the spinal canal, immediately snap-frozen and stored at -80°C.

All animal experiments were conducted in accordance with local authorities (Regierungspräsidium Hannover, Germany, permission numbers: 33–42502-05/963 and 33-42502-04-07/1292).

Histology and immunohistology

Serial transversal sections of formalin fixed, paraffin embedded cervical, thoracic and lumbar spinal cords were stained with haematoxylin and eosin (HE) or Luxol fast blue-cresyl violet (LFB-CV) and semi-quantitatively assessed as previously described [26,30]. Briefly, each section was evaluated for meningitis and leukomyelitis as follows: 0 = no change, 1 = scattered perivascular infiltrates, 2 = 2 to 3 layers of perivascular inflammatory cells, 3 = more than 3 layers of perivascular inflammatory cells, and the degree of demyelination was evaluated as follows: 0 = no change, 1 =<

25%, 2 = 25–50%, 3 = 50–100% of white matter affected. A mean score per mouse was calculated from all three locations evaluated.

Immunohistology was performed on serial sections of the formalin fixed, paraffin embedded spinal cords using the avidin-biotin-peroxidase complex (ABC) method

(Vector Laboratories, Burlingame, CA, USA) with

3,3′-diaminobenzidine-tetrahydrochloride (DAB) as chromogen as previously described [18,26,28,30-32]. The used primary antibodies were directed against glial

fibrillary acidic protein (GFAP, polyclonal rabbit anti-cow, diluted 1:1000, Dako Diagnostika, Hamburg, Germany), myelin basic protein (MBP, polyclonal rabbit anti-human, diluted 1:800, Millipore, Schwalbach, Germany), NG2 (polyclonal rabbit anti-rat, diluted 1:200, Millipore, Schwalbach, Germany), signal transducer and activator of transcription 3 (STAT3, polyclonal rabbit anti-human, diluted 1:400, Acris, Herford, Germany), phosphorylated STAT3 (p-STAT3, monoclonal rabbit anti-human, clone EP2147Y, diluted 1:400, Millipore, Schwalbach, Germany), and TMEV (polyclonal rabbit anti-TMEV capsid protein VP1; diluted 1:2000).

For double-labelling immunohistology, we employed a sequential protocol using a rat-on-mouse alkaline phosphatase (AP) polymer kit (Biocare Medical, Concord, CA, USA), mouse-on-mouse AP polymer kit (Biocare Medical, Concord, CA, USA), or anti-rabbit AP polymer kit (Nichirei Biosciences, Tokio, Japan) with nitrotetrazolium blue chloride / 5-Bromo-4-chloro-3-indolyl phosphate p-toluidine salt as chromogen (NBT/BCIP) for the first primary antibody, followed by the ABC method (Vector Laboratories, Burlingame, CA, USA) with DAB as chromogen for the second primary antibody. Applied first primary antibodies were directed against CD107b (monoclonal rat anti-mouse, clone M3/84 diluted 1:200, AbD Serotec, Oxford, UK), GFAP (monoclonal mouse anti-pig, clone G-A-5, diluted 1:1000, Sigma-Aldrich, Munich, Germany) or NG2 (polyclonal rabbit anti-rat, diluted 1:200, Millipore, Schwalbach, Germany), and the second primary antibody was directed against p-STAT3 (monoclonal rabbit anti-human, clone EP2147Y diluted 1:400, Millipore, Schwalbach, Germany).

Negative control sections were incubated with equally diluted normal rabbit serum (Sigma-Aldrich, Munich, Germany, R9759), a mouse IgG1 anti-isotype antibody (Millipore, Schwalbach, Germany, CBL600), or a rabbit monoclonal anti-human cytokeratin 20 antibody (clone EPR1622Y, diluted 1:400, Millipore, Schwalbach, Germany) instead of the primary antibodies.

The density of GFAP-, NG2-, STAT3-, p-STAT3- and TMEV-positive cells within the spinal cords was counted as described [18,27,28,30]. The MBP-positive white matter area was measured employing the analysis 3.1 software package (SOFT Imaging

system, Münster, Germany) as described [18,26-28,30].

The semi-quantitative scores and cell counts were evaluated for significant differences between TMEV- and mock-infected mice using pair-wise Mann-Whitney U-tests and between lesioned white matter and normal appearing white matter (NAWM) within the TMEV-infected animals using Wilcoxon signed rank tests (IBM SPSS Statistics, Version 20, IBM Corporation, Armonk, NY, USA). Statistical significance was designated as p 0.05.

Microarray analysis

For microarray analysis RNA was isolated from snap-frozen spinal cord specimens using the RNeasy Mini Kit (Qiagen, Hilden, Germany), amplified and labelled employing the MessageAmp II Biotin Enhanced Kit (Ambion, Austin, TX) and hybridized to GeneChip mouse genome 430 2.0 arrays (Affymetrix, Santa Clara, CA) as previously described [30,33]. Background adjustment and quantile normalization was performed using RMAexpress [34]. MIAME compliant data sets are deposited in the ArrayExpress database (E-MEXP-1717; http://www.ebi.ac.uk/arrayexpress).

Gene Set Enrichment Analysis (GSEA), Version 2.0.10 was performed employing Pearson´s correlation coefficient as metric to rank the genes within the gene sets derived from the Gene Ontology (GO) biological process category of the Molecular Signatures Database (MSigDB) Version 3.1 according to their correlation to the mean density of NG2-positive cells per spinal cord as observed by immunohistology [30,35,36]. Orthologous official human gene symbols (HUGO), required as gene identifiers by GSEA were retrieved employing MADGene [37]. Gene sets in a size range  from  10  to  100  genes  with  a  p  ≤  0.05  and  a  false  discovery  rate  q  ≤  0.25  were   selected and ranked according to the normalized enrichment score [36].

Bottom-up analysis of manually selected candidate genes was done using classical statistics as previously described [11,27,38]. Accordingly, 229 candidate genes comprising ligands, receptors, members, activators, inhibitors and downstream targets of the Janus kinase (JAK)-STAT pathway, were manually extracted from the canonical JAK-STAT pathway of the Kyoto Encyclopedia of Genes and Genomes

(KEGG) database (Entry: map04630) and peer-reviewed published literature (Supplemental Table 1) [39]. The normalized expression values of these genes were evaluated for significant differences between TMEV- and mock-infected mice employing independent pair-wise Mann-Whitney U-tests (IBM SPSS Statistics, Version 20, IBM Corporation, Armonk, NY, USA). Statistical significance was designated as p 0.05.

Cell culture

The glial progenitor cell line BO-1 was maintained in vitro as previously described [25,40]. For the proliferation and differentiation studies, cells were seeded into 96-well microtiter plates (Nunc, Wiesbaden, Germany) at a density of 5,000 cells/well and

The glial progenitor cell line BO-1 was maintained in vitro as previously described [25,40]. For the proliferation and differentiation studies, cells were seeded into 96-well microtiter plates (Nunc, Wiesbaden, Germany) at a density of 5,000 cells/well and

Im Dokument Role of JAK-STAT signaling in the pathogenesis of astrogliosis in chronic demyelinating Theilers̉ murine encephalomyelitis (Seite 39-0)