Transplanted neural precursor cells reduce brain inflammation to attenuate chronic experimental autoimmune encephalomyelitis

Experimental Neurology 198 (2006) 275 – 284 www.elsevier.com/locate/yexnr

Transplanted neural precursor cells reduce brain inflammation to attenuate chronic experimental autoimmune encephalomyelitis Ofira Einstein a , Nikolaos Grigoriadis b , Rachel Mizrachi-Kol a , Etti Reinhartz a , Eleni Polyzoidou b , Iris Lavon a,c , Ioannis Milonas b , Dimitrios Karussis a , Oded Abramsky a , Tamir Ben-Hur a,⁎ a

Department of Neurology, The Agnes Ginges Center for Human Neurogenetics, Hadassah - Hebrew University Hospital, Jerusalem, Israel b B’ Department of Neurology, University of Thessaloniki, Greece c Leslie and Michael Gaffin Center for Neuro-Oncology, Hadassah-Hebrew University Hospital, Jerusalem Received 17 February 2005; revised 6 September 2005; accepted 8 November 2005 Available online 10 February 2006

Abstract Stem cell transplantation was introduced as a mean of cell replacement therapy, but the mechanism by which it confers clinical improvement in experimental models of neurological diseases is not clear. Here, we transplanted neural precursor cells (NPCs) into the ventricles of mice at day 6 after induction of chronic experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). Transplanted cells migrated into white matter tracts and attenuated the clinical course of disease. NPC transplantation down-regulated the inflammatory brain process at the acute phase of disease, as indicated by a reduction in the number of perivascular infiltrates and of brain CD3+ T cells, an increase in the number and proportion of regulatory T cells and a reduction in the expression of ICAM-1 and LFA-1 in the brain. Demyelination and acute axonal injury in this model are considered to result mainly from the acute inflammatory process and correlate well with the chronic neurological residua. In consequence to inhibition of brain inflammation, precursor cell transplantation attenuated the primary demyelinating process and reduced the acute axonal injury. As a result, the size of demyelinated areas and extent of chronic axonal pathology were reduced in the transplanted brains. We suggest that the beneficial effect of transplanted NPCs in chronic EAE is mediated, in part, by decreasing brain inflammation and reducing tissue injury. © 2005 Elsevier Inc. All rights reserved. Keywords: Neural stem cells; Transplantation; Inflammation; Demyelination; Multiple sclerosis

Introduction Neural (stem) cell transplantation has been proposed as a means of cell replacement therapy for diseases of the central nervous system (CNS). Transplanted neural precursors of various origins improved the clinical outcome in experimental models of stroke (Veizovic et al., 2001), spinal cord trauma (McDonald et al., 1999) and multiple sclerosis (MS) (Pluchino et al., 2003). Although transplanted myelin-forming cells have remarkable remyelinating properties, resulting in restoration of electrophysiological nerve function (Milward et al., 1997), the mechanisms by which transplanted cells exert a beneficial ⁎ Corresponding author. Department of Neurology, Hadassah Hebrew University Medical Center, Ein-Kerem, PO Box 12,000, Jerusalem 91120, Israel. Fax. +972 2 6437782. E-mail address: [email protected] (T. Ben-Hur). 0014-4886/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2005.11.007

clinical effect in chronic experimental autoimmune encephalomyelitis (EAE), the animal model of MS, are not clear. Cellmediated immunity against myelin antigens, inducing demyelination and axonal injury, is a major factor in the pathogenesis of MS and EAE. In MS, the general failure of endogenous remyelination (Franklin, 2002) and inflammation-related axonal pathology (Kornek et al., 2000; Trapp et al., 1998) lead to accumulated neurological disability (Ferguson et al., 1997; Lovas et al., 2000). We have recently shown that neurosphere transplantation inhibited the clinical and pathological features of acute EAE, a model for disseminated brain inflammation with a minor demyelinating component (Einstein et al., 2003). We therefore hypothesized that the beneficial effects of transplanted cells in chronic EAE may be mediated, in part, by attenuation of the inflammatory brain process, leading to reduction in tissue injury. Here, we transplanted NPCs intracerebroventricularly (ICV) into C57Bl/6 mice with the

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MOG35–55 EAE model. In this model, the acute inflammationmediated demyelination and axonal injury lead to a chronic clinical course of disability. We examined whether transplanted precursor cells inhibit the acute detrimental inflammatory brain process, leading to reduced demyelination and axonal injury and to improvement in the clinical outcome. Materials and methods Growth of mouse neurospheres Cerebral hemispheres were dissected from newborn C57Bl/6 mice (and from green fluorescent protein, GFP+ transgenic mice, “green mouse FM131”, courtesy of M. Okabe, Osaka, Japan). Following removal of meninges, the tissue was minced, digested in 0.025% trypsin for 20 min and dissociated with a 5ml pipette into a single cell suspension. The cells were suspended in serum-free F12/DMEM medium supplemented with 10 mg% human apo-transferin, 1 mM sodium-pyruvate, 0.05% BSA, 10 ng/ml D-biotin, 30 nM sodium–selenite, 20 nM progesterone, 60 μM putrescine, 25 μg/ml BSA, 2 mM L-glutamine and 25 μg/ml gentamycin (all from Sigma). The cells were plated 10 × 106 cells/T-75 uncoated flask and supplemented with 10 ng/ml of basic fibroblast growth factor (FGF2, R&D) and 20 ng/ml of epidermal growth factor (EGF, R&D), added daily. In these conditions, most cells died and approximately 0.2% of cells proliferated into clusters of small round cells that grew into floating spheres containing multipotential NPCs. EAE induction and neurospheres transplantation Animal experimentation received the approval of institute's Ethics Committee and was conducted in accordance with the United States Public Health Service Policy on Human Care and Use of Laboratory Animals. EAE was induced in 6–7 week old female C57B/6 mice by immunization with an emulsion containing 300 μg of purified myelin oligodendrocyte glycoprotein (MOG) peptide (MEVGWYRSPFSRVVHLYRNGK, corresponding to residues 35–55) in PBS and an equal volume of complete Freund's adjuvant containing 5 mg H37RA (Difco). 0.2 ml of the inoculum was injected subcutaneously at day of induction (day 0) and at day 7. In addition, 300 ng of Bordetella pertusis toxin (Sigma) in 0.2 ml PBS was injected intraperitoneally at day of induction and at day 2. Clinical signs of EAE appeared typically after 12–14 days, reaching to the maximum score after 5–6 days with no recovery. The animals were followed clinically for up to 80 days and were scored daily for neurological symptoms as follows: 0, asymptomatic; 1, partial loss of tail tonicity; 2, atonic tail; 3, hind leg weakness and/or difficulty to roll over; 4, hind leg paralysis; 5, four leg paralysis; 6, death due to EAE. Six days post-EAE induction, mice were anesthesized with intraperitoneal injection of pentobarbital (0.6 mg/10 g) and were fixed in a stereotactic device. Quantities of 2.5 × 103 spheres (GFP+ or after 72 h incubation with 25 μg/ml Bromodeoxyuridine, BrdU; ∼100 cells/sphere; n = 34) or dead 2.5 × 103 spheres (obtained by 3 cycles of freezing and

thawing, n = 8) or 2.5 × 105 GFP+ astrocytes (n = 12) or F12/ DMEM medium (n = 31) in a volume of 5 μl were injected once into each lateral ventricle (ICV transplantation). Brain fixation and histological preparation EAE animals were sacrificed for histopathological analysis at 2 time points: at peak of first relapse (day 18 post-EAE induction; neurosphere-transplanted n = 10, astrocyte-transplanted n = 7, control n = 10), to observe the pathological changes at the acute phase, and at the end of the follow-up period (day 80 post-EAE induction; neurosphere-transplanted n = 11, control n = 10), to observe the pathological changes at the chronic phase. Animals were anesthetized with a lethal dose of pentobarbital and brains and spinal cords were perfused via the ascending aorta with ice-cold PBS followed by cold 4% paraformaldehyde in PBS. The tissues were dissected and postfixed by immersion in the same fixative for 24 h at 4°C. Tissues were deep freezed in liquid nitrogen and serial 6–8 μM brain axial and longitudinal frozen sections and spinal cord longitudinal frozen sections were performed. Immunofluorescent staining of NPCs in vitro and in vivo Mouse IgM anti-PSA-NCAM (generous gift of G. Rougon, dilution 1:200), mouse IgG anti-nestin (clone 401, 1:50, Chemicon), rabbit IgG anti-NG2 (1:50, Chemicon), rabbit anti-galactocerebroside (GalC, 1:20, Chemicon), mouse IgM anti-O4 (1:20, Chemicon), rabbit anti-glial fibrillary acidic protein (GFAP, 1:100, Dako) and mouse IgG anti-neuronal nuclei (NeuN, 1:50, Chemicon) were used, followed by Texas red- or Alexa 488-conjugated goat anti-mouse IgM (1:100, Jackson, West Grove, PN), goat anti-rabbit IgG (1:100, Molecular Probes) or goat anti-mouse IgG (1:100, Molecular Probes) as secondary antibodies, where appropriate. For in vitro characterization, floating spheres were adhered to 35 mm tissue culture dishes coated with 10 μg/ml poly-D-lysine and 5 μg/ml fibronectin (Sigma) for 2 h to stain for PSA-NCAM and nestin. Other cultures were stained after 1 or 5 days of differentiation after withdrawal of FGF2 and EGF for NG2, O4, GalC, GFAP and NeuN. Cell surface markers were stained in living cells followed by fixation in 95% ethanol–5% acetic acid for 10 min in −20°C and then staining of intracellular antigens. The cells were incubated with primary antibody for 45 min followed by 30 min incubation with a secondary antibody. BrdU- immunofluorescence and double staining in vivo were performed on 6– 8 μm axial frozen brain sections at 2.85 mm below the bregma. Sections were fixated in acetone (for 10 min at −20°C), dried and then incubated in 0.3% H2O2 in methanol for 15 min. After PBS washes, the sections were incubated in 0.1 mg/ml proteinase K for 15 min. The sections were treated with 4 N HCl for 10 min and washed to neutralize the pH. Sections were incubated with anti-BrdU (clone Bu20a, Dako, 1:20 dilution) overnight at 4°C. A goat anti-mouse IgG secondary antibody, conjugated to Alexa 488, was added for 50 min at room temperature. Fluorescent GFP+ cells were detected in axial brain frozen sections at 2.85 mm below the bregma.

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Quantification of the brain inflammatory process The evaluation of the inflammatory process was performed at 18 days post-EAE induction, representing the acute phase of disease, by using several parameters. Hematoxylin–eosin (H&E) staining was performed on three serial axial frozen sections at level of 2.35 mm below bregma in order to detect perivascular mononuclear infiltrations. A blood vessel surrounded by 10 nuclei or more was considered as a positive perivascular infiltrate. The total average number of infiltrations per mm2 in the corpus callosum and in the cerebellar white matter was counted. The expression of intercellular adhesion molecule-1 (ICAM-1) and leukocyte function antigen-1 (LFA1) in the brain was examined on three serial axial frozen sections at 2 mm and 2.6 mm below bregma, respectively, using immunohistochemical stainings. Sections were post-fixed in acetone and anti-mouse CD11a (ICAM-1, clone 121/7, SouthernBiothech, 1:200 dilution) and rat anti-mouse CD54 (LFA-1, clone KAT-1, Serotec, 1:200 dilution) antibodies were incubated for 1.5 h followed by biotinylated anti-mouse secondary antibody and ABC kit (Vectastain) and developed with 3,3′diaminobenzidine DAB (Sigma). Five microscopic fields (at ×10 magnification) spanning the corpus callosum and five microscopic fields spanning the cerebellar white matter (at ×10 magnification) were obtained under fixed illumination from each section and stored on a computer using a video camera. The stained perivascular area was measured in pixels by using the Sigma Scan-pro software, and presented as the percentage (average ± SD) of the area of corpus callosum or cerebellar white matter. Immunofluorecent stainings for CD3 T cells, CD25, CD25+/CD62L+ and NK-T regulatory cell subtypes were performed on three longitudinal brain serial frozen sections for each cell subtype, at 1, 1.25, 1.55 and 1.85 mm lateral to the bregma, respectively. Sections were incubated for 2 h in room temperature with rat anti-CD3 (clone CD3-12, Serotec, dilution 1:400) to detect CD3+ T cells, anti-IL-2Rα rabbit polyclonal antibody (CD25, clone M-19, Santa Cruz, dilution 1:500) alone or together with rat anti-L-selectin monoclonal IgG antibody (CD62L, clone Mel-14, Santa Cruz, dilution 1:150) to detect CD25+ or CD25+/CD62+ cells, respectively, or rabbit anti-asialo GM1 (Wako, dilution 1:150) together with hamster anti-mouse TCR α/β (clone H57-597, Serotec, dilution 1:150) to detect NK-T cells. A goat anti-rabbit IgG secondary antibody for CD25 and asialo GM1 and a goat anti-rat IgG secondary antibody for CD62L conjugated to Alexa-488 (NBT, 1:100 dilution) were added for 50 min in room temperature. The total average number per mm2 of each cell type was counted. Quantification of axonal damage Acute axonal damage was evaluated at day 18 post-EAE induction, using amyloid precursor protein (APP) immunohistochmistry and Bielschowsky silver stain, which was also used to evaluate chronic axonal pathology at 80 days post-EAE induction. Bielschowsky silver stain for axons was performed on five longitudinal brain and spinal cord serial sections

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according to standard procedure. Axonal damage evaluation was performed on five microscopic fields (at ×40 magnification) throughout the corpus callosum, cerebellum, brain stem and spinal cord, separately, based on axonal density, the presence of axonal bulbs, spheroids, ovoids, dilated or dystrophic axons and was assessed according to a rating score as follows: 0, normal morphology and density; 1, 1–5 pathological axons; 2, 6–10 pathological axons or 10–20% axonal loss; 3, 6–10 pathological axons and 10–20% axonal loss; 4, N10 pathological axons or 20–50% axonal loss; 5, 6–10 pathological axons and 20–50% axonal loss. APP immunohistochemistry was performed on five longitudinal brain and spinal cord serial sections (0.75 mm lateral to the bregma) in order to detect APP accumulation within acutely injured axons. Sections were incubated with an anti-APP (c-terminus) polyclonal antibody (Sigma, dilution 1:1000) for 2 h followed by biotinylated anti-rabbit secondary antibody and ABC kit (Vectastain) and developed with DAB (Sigma). APP accumulation was assessed on five microscopic fields per section (at ×40 magnification) throughout corpus callosum, cerebellum, brain stem and spinal cord, separately. All sections were studied under fixed illumination of a light microscope and were evaluated by using a scale specifically created for this purpose based on accumulation of APP: 0, no staining at all; 1, 1–5 APP + axons; 2, 6–10 APP+ axons; 3, N10 APP+ axons. On each microscopic field of APP and Bielschowsky silver staining, the presence of perivascular mononuclear infiltrates was examined, using hematoxylin counterstain for nuclei. The fraction of microscopic fields in which axonal injury was associated with a local inflammatory infiltrate was calculated. Quantification of myelin destruction Detailed evaluation of myelin destruction was performed at 18 days post-EAE induction. Brains were dissected to isolate the corpus callosum and periventricular area. These segments were cut using a vibratome, oriented to permit 50 μm transverse sections of the axons and embedded in resin. Corpus callosum of each brain was cut to serial semithin sections (1 μm) and stained with 1% toluidine blue (Sigma). From each brain, five randomly chosen semithin sections were picked. Three microscopic fields per each section were photographed with a light microscope (×100). The intactness of myelin was evaluated morphologically, based on color intensity and thickness of myelin. The numbers of normal myelinated, demyelinated and thin, remyelinated fibers were counted. The mean fraction of demyelinated and remyelinated axons from the total number of axons per microscopic field was then calculated. At 80 days post-EAE induction, chronic myelin destruction was evaluated by Kluver Barerra histochemistry on five longitudinal brain sections (0.75 mm lateral to the bregma). Each brain section was visualized under fixed illumination of a light microscope (at ×10 magnification) and unstained area, representing areas of myelin destruction, and was stored on a computer using a video camera. The unstained area was measured in pixels by using the Sigma Scan-pro software, and presented as the percentage (average ± SD) of the area of the hemispheres.

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RT-PCR analysis Total RNA was prepared using SV total RNA kit (Promega, Madison, WI). cDNA was prepared from 2 μg of total RNA using MuLV reverse transcriptase (Applied Biosystems) and random Hexamers according to manufacturer's instructions. Reaction mixture included 1 μg of cDNA, 300 nM concentrations of the appropriate forward and reverse primers (Syntezza) and 7.5 μl of the master mix buffer containing nucleotides, Taq polymerase (ABgene), in a total volume of 15 μl. Gene amplification was carried out using the GeneAmp 9700 PCR system (Applied Biosystems). Amplification included one stage of 10 min at 95°C followed by 35 cycles of two-step loop: 15 s at 95°C and 1 min at 60°C. The following primers were used with the expected PCR product length in base pair (bp): β-actin: TTG TAA CCA ACT GGG ACG ATA TGC(+), GAT CTT GAT CTT CAT GAT GCT AGG(−); insulin like growth factor-1 (IGF-1): GCG CTC TGC TTG CTC ACC T(+), TCG GTC CAC ACA CGA ACT GA(−); nerve growth factor (NGF): ACC CAC CCA GTC TTC CAC ATG(+), CCT CCT TGC CCT TGA TGT CTG(−); brain-derived neurotrophic factor (BDNF): GAG CAC GTC ATC GAA GAG CTG (+), TGA GCA TCA CCC GGG AAG T(−); glial-derived neurotrophic factor (GDNF): TTG CAG CGG TTC CTG TGA AT(+), CAT GCC TGG CCT ACT TTG TCA(−); neurotrophin 3 (NT3): TCA CCA CGG AGG AAA CGC TA(+), TGG CTG AGG ACT TGT CGG TC(−); ciliary neurotrophic factor (CNTF): AAG AGA GGG AGA AGG CGA AAA G(+), CAC ATC CTT CCA TCT CAC AAC G(−); leukemia inhibitory factor (LIF): CGT GGA AAA GCT ATG TGC GC(+), TGC GAC CAT CCG ATA CAG C(−) and erythropoietin (EPO): GAG GTG GAA GAA CAG GCC ATA GA(+), TGG TGG CTG GGA GGA ATT G(−). Statistical analysis All quantitative pathological evaluations were made in a uniform fashion by an observer who was blinded to the experimental group. Quantitated clinical and pathological data from several experiments were pooled to obtain the mean ± SD or SE for each experimental group. For each parameter, transplanted and control groups were compared using onetailed Student's t test or the Mann–Whitney test. Statistical analysis for the clinical data of the various experimental groups was performed by 2-way ANOVA, followed by Bonferroni post hoc test. Differences were considered significant at P b 0.05. Results Expansion of newborn mouse neurospheres Multipotential NPCs were isolated from newborn C57Bl/6 mouse cerebral hemispheres and expanded in floating spheres as nestin+, PSA-NCAM+ cells (Figs. 1A–B). These spheres contain only rare (b1% of cells) GFAP+ astrocytes or NG2+ oligodendrocyte progenitors. The potential of the spheres to

generate differentiated cells of neural lineages was examined in vitro following plating on polylysine and fibronectin and removal of growth factors. Within 1 day, multiple NG2+ oligodendrocyte progenitor cells (17 ± 3.5%) were observed migrating from the spheres (Fig. 1C). After 5 days of differentiation, the spheres generated 29 ± 3% O4+ oligodendrocytes, 31 ± 6.4% GalC+ oligodendrocytes, 45 ± 6.4% GFAP+ astrocytes and 3 ± 1.5% NeuN+ neurons (Figs. 1D–E). Neurospheres were passaged by dissociation into a single cell suspension, from which new spheres were grown. Secondary and tertiary spheres generated the 3 neural lineages as above, demonstrating the multipotentiality and self-renewal qualities of the precursor cells. To facilitate the identification of sphere cells in host brain after transplantation, we tagged them with BrdU or grew the neurospheres from transgenic mice expressing GFP. Integration of transplanted cells in the host EAE brain and attenuation of clinical disease We have previously shown in acute EAE in Lewis rats that brain inflammation attracts transplanted cells to migrate into the brain parenchyma along white matter tracts (Ben-Hur et al., 2003). We now transplanted 2.5 × 103 undifferentiated spheres (corresponding to ∼250,000 cells) into both lateral ventricles of C57Bl/6 mice at 6 days post-induction of chronic EAE. The transplanted cells disseminated along inflamed white matter tracts. At 12 days post-grafting, numerous transplanted cells were found in the corpus callosum, internal and external capsule and other periventricular white matter tracts in association with the inflammatory process (Figs. 1F–I). The transplanted cell density at 12 days post-transplantation in these areas was 17.79 ± 1.07 BrdU+ cells/mm2 (23.7 ± 1.8/mm2 in the corpus callosum) and 16.16 ± 1.51 GFP+ cells/mm2 (26.5 ± 1.24/mm2 in the corpus callosum). Double fluorescence immunostaining indicated that only a fraction of the transplanted cells acquired glial specific markers at this stage. NG2 proteoglycan, a marker of oligodendrocyte progenitor cells, was expressed by 26 ± 3% of the transplanted GFP cells (Fig. 1J). Also, 9 ± 1% of the grafted cells expressed the oligodendrocyte marker GalC+ (Fig. 1K) and 11 ± 2% expressed the astrocytic marker GFAP (Fig. 1L). At 74 days post-transplantation, there were numerous transplanted GFP+ cells disseminated throughout the corpus callosum and white matter tracts (Fig. 1M). The transplanted cell density at 74 days post-transplantation in deep cerebral white matter tracts was 18.24 ± 1.33 GFP+ cells/mm2. At this stage, 17 ± 2% of GFP+ cells expressed the GalC oligodendrocyte marker. Transplant-derived neurons were not found in the corpus callosum and periventricular white matter. The clinical score was evaluated daily in individual mice and the cumulative severity of disease (“area under curve”) was calculated at day 18 post-EAE induction (peak of the acute relapse) and at the end of the follow-up (day 80, representing the chronic phase). A mild decrease in disease severity by neurosphere transplantation was noted already in the acute phase. The most significant attenuation of the clinical paralytic signs was observed in the chronic phase (Table 1, Fig. 2).

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Fig. 1. Characterization of neurospheres in vitro and in vivo. Neurosphere cells stained for nestin (A) and PSA-NCAM (B). Numerous NG2+ cells appeared after 1 day of differentiation in vitro (C). After 5 days of differentiation, the spheres generated mainly O4+ oligodendrocytes (red) and GFAP+ astrocytes (green) (D) and only few NeuN+ neurons (E). During the acute phase (12 days post-transplantation) transplanted undifferentiated sphere cells migrated into inflamed white matter tracts in the brain. Perivascular infiltrates (arrows) in the corpus callosum (F) attracting transplanted BrdU+ cells (G, adjacent section). BrdU+ cells were also found in more distant white matter tracts, such as the external capsule (H—Dapi counterstain, I—BrdU). Confocal microscopy of immunofluorescent stained sections containing transplanted GFP+ cells indicated that a fraction of the cells acquired glial lineage markers (red), including the progenitor marker NG2 (J), GalC (K) and GFAP (L). GFP fluorescence showed that transplanted cells persisted in the host brain during the chronic phase (M, corpus callosum; 74 days post-transplantation). Scale bars: A– E, J–L = 10 μm; F–G, M = 100 μ; E–I = 50 μ.

Reduction of brain inflammation by neurosphere transplantation Since the chronic clinical and pathological manifestations of MOG35–55 EAE in C57Bl/6 mice are caused mainly by the inflammatory process during the acute phase of the disease, we investigated whether the beneficial effect of neurosphere transplantation is related to inhibition of tissue inflammation at 18 days post-EAE induction. The numbers of perivascular immune cell infiltrations and CD3+ T cells were significantly decreased in the brains of transplanted mice, compared with that in controls (Table 2, Figs. 3A–D). In the corpus callosum of

transplanted mice, there were 35% less infiltrates and 20% less CD3+ T cells than in non-transplanted mice. ICAM-1 and its ligand, LFA-1, play a central role in the transendothelial migration of leukocytes to the CNS in EAE and MS and may serve as markers of active inflammation (Cotran and MayadasNorton, 1998; Van de Stolpe and van der Saag, 1996; YusufMakagiansar et al., 2002). To further examine the notion that neurosphere transplantation may reduce detrimental brain inflammation, we examined the perivascular expression of ICAM-1 and LFA-1 in the brain by immunohistochemistry. Quantification by computer-assisted image analysis showed that ICAM-1 and LFA-1 expression was reduced by 23% and

Table 1 Neurosphere-transplanted chronic EAE mice exhibit a milder course of disease than control (medium-injected) EAE mice by all 3 clinical parameters Clinical parameters Acute phase (day 18)

Clinical score Maximal clinical score Cumulative disease score

Chronic phase (day 80)

Transplanted

Control

P value

Transplanted

Control

P value

2.43 ± 1.47 (n = 31) 2.51 ± 1.49 (n = 31) 10.46 ± 6.83 (n = 31)

2.97 ± 1.41 (n = 34) 3 ± 1.38 (n = 34) 13.45 ± 6.91 (n = 34)

0.07 0.09 0.042

2.33 ± 2.05 (n = 21) 3.42 ± 1.45 (n = 21) 166.21 ± 120.2 (n = 21)

3.72 ± 1.93 (n = 18) 4.36 ± 1.61 (n = 18) 240.63 ± 124.01 (n = 18)

0.018 0.032 0.032

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Fig. 2. Neurosphere transplantation ameliorates the clinical severity of chronic EAE.

30%, respectively, in the corpus callosum of sphere-transplanted brains, as compared with that in sham-injected EAE brains (Table 2, Figs. 3E–H). MOG35–55 induced EAE is a CD4+ Th1-mediated autoimmune disease in which autoreactive T cells specific for myelin components enter the CNS, initiating a cascade of inflammation and demyelination (Mendel et al., 1995). Several lines of evidence suggest that the progression of the autoimmune process is controlled by regulatory T cells, which block and down-regulate self-reactive lymphocytes. CD4+/ CD25+ T cells and NK-T cells (characterized by the expression of NK cell markers along with a restricted α–β TCR) are two subsets of the regulatory T cell arsenal that suppress EAE (Kohm et al., 2002; Mars et al., 2002; Shevach, 2002). Regulatory T cells can be identified in the brain as CD25+/ CD62L+ cells and NK-T cells as asialo-GM1+/TCR+ cells. To further substantiate the effect of neurosphere transplantation on the autoimmune process in the brain, we examined the presence of these regulatory cells in the brains of transplanted versus control EAE mice at day 18 post-EAE induction. In transplanted mice, there was a significant increase in the number of CD25+ and CD62L+/CD25+ cells (Table 2, Figs. 3I–L). In nontransplanted animals, there were 1.6 ± 0.36 CD25+ cells per mm2. In transplanted animals, the average CD25+ cell density increased more than 2-fold to 3.3 ± 0.6 per mm2 (P = 0.001). Thus, the ratio of CD25+ cells to CD3+ cells increased by 2.5fold in transplanted mice. A similar increase in the number of

asialo-GM-1+/TCR+ NK-T cells in transplanted mice was also observed, but was not statistically significant, probably due to their low number (Table 2). Recent studies have shown that several neurotrophic factors may inhibit brain inflammation in general and EAE in particular (Butzkueven et al., 2002; Hammarberg et al., 2000; Li et al., 2004; Linker et al., 2002; Lovett-Racke et al., 1998; Villoslada et al., 2000). Neural stem cells can produce neurotrophic factors that have functional effects on axonal growth in vivo (Lu et al., 2003). We therefore examined whether our neurosphere cells express the genes of the various neurotrophic factors that were shown to possess an inhibitory effect on brain inflammation. RT-PCR showed that NPCs express IGF-1, NGF, BDNF, GDNF, NT3, CNTF, LIF and EPO (Fig. 3M). The demyelinating process and axonal injury are reduced in neurosphere-transplanted brains The autoimmune inflammatory brain process is the underlying cause of demyelination and axonal pathology in EAE and MS. We therefore examined whether modulation of brain inflammation by transplanted cells was associated with reduction in tissue injury at the acute phase (18 days postEAE induction). To quantify the demyelinating process, we performed toluidine-blue stainings on semithin longitudinal sections of the corpus callosum of transplanted (n = 6) and nontransplanted (n = 5) EAE brains. Fluorescence microscopy on vibratome sections identified the presence of multiple GFP+ cells in the corpus callosum of transplanted brains prior to preparation of the semithin sections. Nude, demyelinated axons and thinly remyelinated axons were identified by their typical morphology. In sham-transplanted chronic EAE mice, there were 3.7 ± 0.16% demyelinated axons and 2.7 ± 0.19% remyelinated axons (Fig. 4A). In transplanted mice, there were 2.5 ± 0.25% demyelinated axons and 3.1 ± 0.3% remyelinated axons (Fig. 4B). Thus, neurosphere transplantation caused a 32.4% reduction in the primary demyelinating process (P b 0.001) and only a mild, albeit significant 12.9% increase in the remyelinating process (P = 0.007). The axonal pathology in EAE and MS is also related to the severity of inflammation (Bitsch et al., 2000). The

Table 2 Transplanted neurospheres modulate the inflammatory brain process Inflammatory parameters (acute phase) Neural sphere transplantation 2

Perivascular infiltrates (per mm ) CD3 (per mm2) ICAM-1 (% per brain area) LFA-1 (% per brain area) CD25 (per mm2) CD25, CD62L (per mm2) NK-T (per mm2)

Corpus callosum Cerebellum Hemispheres Corpus callosum Cerebellum Corpus callosum Cerebellum Hemispheres Hemispheres Hemispheres

Transplanted

Control

P value

1.8 ± 0.31 (n = 8) 3.09 ± 0.93 (n = 8) 9.74 ± 1.75 (n = 8) 5.17 ± 0.02% (n = 5) 6.1 ± 0.58% (n = 5) 4.7 ± 1.2% (n = 5) 5.5 ± 0.6% (n = 5) 3.3 ± 0.6 (n = 5) 0.63 ± 0.11 (n = 5) 0.24 ± 0.0.6 (n = 5)

2.74 ± 0.9 (n = 7) 4.14 ± 0.43 (n = 7) 11.84 ± 2.29 (n = 6) 6.7 ± 0.43% (n = 6) 8.1 ± 0.9% (n = 6) 6.7 ± 0.69% (n = 5) 6.4 ± 0.7% (n = 5) 1.6 ± 0.36 (n = 5) 0.44 ± 0.13 (n = 7) 0.2 ± 0.04 (n = 5)

0.016 0.013 0.037 0.016 0.05 0.009 0.047 0.0014 0.011 0.11

Quantification of the inflammatory process at the acute phase showed that neurosphere transplantation decreased the numbers of perivascular infiltrates and CD3+ T cells, inhibited the expression of ICAM-1 and LFA-1 and increased the number of CD25+ and CD25+/CD62L+ regulatory cells in EAE brains.

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Fig. 3. Attenuation of the acute inflammatory process of chronic EAE by neurosphere transplantation. H&E staining, CD3 immunofluorescent, ICAM-1 and LFA-1 immunohistochemistry were performed in neurosphere-transplanted brains (A, C, E, G, respectively) and in medium-injected EAE brains (B, D, F, H, respectively). There were significantly less infiltrates and CD3+ T cells and ICAM-1 and LFA-1 expressions were reduced in sphere-transplanted brains. CD25+ cells were found in inflammatory infiltrates of EAE brains (I–L). Sphere-transplanted EAE brains contained significantly more CD25+ cells (I, J—Dapi counterstain) than mediuminjected brains (K, L—Dapi counterstain). Neurosphere cells express multiple neurotrophic factor genes, as indicated by RT-PCR (M). Control—no cDNA. Scale bars: A, B: 100 μ, C–L: 50 μ.

accumulation of APP in axons is a reliable marker for acute axonal injury (Ferguson et al., 1997). Quantification of the numbers of APP+ axons indicated a significant 56 ± 6% reduction in axonal injury (ranging from 48% to 61% in different brain regions) in transplanted mice brains in the acute phase (Figs. 4C–D, Table 3). Axonal injury and loss may also be visualized as spheroids and premature endings of axons and by reduced axonal density using Bielschowsky's stain. In transplanted mice, the axonal pathology, as determined by Bielschowsky staining, was reduced by 51 ± 23% (range: 22– 76% in various brain regions) in the acute phase. We next examined whether the modulated brain inflammatory process in transplanted animals was associated directly with a local reduction in axonal damage. To this end, we examined the presence of axonal pathology in microscopic fields which contained inflammatory infiltrates. In sham-transplanted mice (n = 7), axonal pathology was found in 88.1 ± 3.05% of microscopic fields containing infiltrates (Fig. 4E). In comparison, in transplanted mice (n = 5), axonal pathology was found only in 63.34 ± 4.31% of microscopic fields that exhibited inflammation (28% reduction, P b 0.01, chi square = 18.9, df = 3). Thus, in transplanted mice, there was a 3-fold increase in the fraction of microscopic fields in which inflammatory process was not associated directly with axonal pathology (Fig. 4F). In conclusion, neurosphere transplantation attenuated the brain inflammatory process and reduced the extent of demyelinating process and of acute axonal injury.

Tissue pathology at the chronic phase of disease represents the final outcome of the inflammation-mediated acute disease process and is correlated to the chronic neurological sequela of disease. We therefore compared the extent of chronic tissue pathology between transplanted and non-transplanted EAE mice. Demyelination was quantified by computerized image analysis of Kluver Barrera stained brain sections. In transplanted mice, the areas of demyelination were reduced by 79.5% as compared with control chronic EAE mice (Figs. 4G– H, Table 3). In transplanted mice, the chronic axonal pathology, as determined by Bielschowsky staining, was reduced by 44 ± 5% (range: 39–53%) as compared to control EAE mice (Table 3). Astrocytes and dead NPCs do not attenuate chronic EAE The controls, dead neurosphere cells (n = 8 mice) or purified primary astrocytes (n = 12 mice), both derived from GFP transgenic mice, were transplanted intraventricularly on day 6 post-EAE induction. While neurosphere-transplanted mice faired significantly better than all other experimental groups, there was no difference in the clinical severity of chronic EAE between mice transplanted with astrocytes, dead neurospheres and medium (not shown). Fluorescence microscopy indicated that astrocytes did not migrate from the ventricular space to the brain parenchyma and that the transplanted dead cells had been cleared from the brain. There was no difference in the number of

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Fig. 4. Attenuation of the primary demyelinating process and inflammation-associated acute axonal injury by neurosphere transplantation. Toluidine-blue stained semithin sections at the acute phase (18 days post-EAE induction) enabled the quantification of the percentage of nude, demyelinated axons and of remyelinated axons. In transplanted EAE brains (B), there were significantly less demyelinated axons (arrowheads) and more remyelinated axons (arrows) than in non-transplanted EAE brains (A). APP staining at the acute phase showed that in sphere-transplanted brains (D) there was less axonal injury than in medium-injected EAE brains (C; arrows—APP+ axons). Bielschowsky staining at the acute phase showed multiple spheroids (arrows), especially around inflammatory infiltrates in mediuminjected brains (E). In sphere-transplanted brains, there was a significantly higher proportion of infiltrates that were not associated with axonal pathology (F). Kluver Barrera staining at the chronic phase (80 days post-EAE induction) showed that there was less demyelination in sphere-transplanted brains (H) than in medium-injected brains (G). Scale bars: A–B = 5 μ; C–D = 100 μ; E–F = 50 μ, G–H = 1 mm.

inflammatory infiltrates between astrocyte-transplanted and sham-transplanted mice. In the corpus callosum, there were 2.6 ± 0.59 perivascular infiltrates per mm2 in transplanted mice (n = 7) versus 2.58 ± 0.26 per mm2 in control EAE mice (n = 6, P = 0.46). In the cerebellum, there were 5.08 ± 0.62 and 5.03 ± 0.33 infiltrates per mm2, respectively (P = 0.43). This further supports the notion that the beneficial effect of transplanted NPCs is related to attenuation of the inflammatory process. Discussion The primary autoimmune process in the C57Bl/6 MOG35– 55 EAE model is directed against MOG, a myelin antigen, and causes demyelination and axonal injury, mainly during the acute phase of disease. It was shown that the chronic neurological impairment in EAE mice is best correlated to the chronic axonal pathology (Steinman, 2001; Wujek et al., 2002). Here, we showed that intraventricularly transplanted NPCs integrate into white matter tracts of the brain and attenuate chronic EAE. Neurosphere transplantation attenuated the acute, detrimental inflammatory process, as indicated by down-regulation of cellular infiltration (and specifically of CD3+ T cells), downregulation of ICAM-1 and LFA-1 expression and up-regulation

in the ratio of CD25+ and CD25+/CD62L+ regulatory cells in the transplanted brains. Consequently, transplanted brains exhibited a reduction in both the demyelinating process and axonal injury that occur in the acute phase. There was a 32% decrease in the number of demyelinated axons in the transplanted brains and a 28% reduction in inflammationassociated acute axonal injury. Finally, the area of demyelination and extent of axonal pathology were reduced in the transplanted brains in the chronic phase, consistent with their improved clinical outcome. The exact mechanism by which transplanted NPCs attenuate brain inflammation is not clear yet. We have recently shown that NPCs have a direct inhibitory effect on the proliferation of EAE-derived lymphocytes in response to MOG and to ConA when co-cultured in vitro (Einstein et al., 2003). Neural precursors produce a variety of neurotrophic factors (Lu et al., 2003) that have both neuroprotective and immunomodulatory functions. Several neurotrophins inhibit EAE by enhancing oligodendrocyte survival (Butzkueven et al., 2002; Linker et al., 2002) and by decreasing neuroinflammation (Flugel et al., 2001; Hammarberg et al., 2000; Lovett-Racke et al., 1998; Villoslada et al., 2000; Zhang et al., 2005). We have also found the expression of NGF, BDNF, CNTF, LIF, IGF-1, EPO, NT3 and GDNF in our neurosphere cells. These neurotrophic factors

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Table 3 Neurosphere transplantation reduces demyelination and axonal pathology in chronic EAE Axonal pathology and demyelination parameters APP (acute phase)

Transplanted

Control

P value

Corpus callosum

0.38 ± 0.04 (n = 5)

0.979 ± 0.073 (n = 7)

b0.0001

Cerebellum

0.81 ± 0.062 (n = 5)

1.566 ± 0.101 (n = 7)

b0.0001

Brain stem

0.61 ± 0.068 (n = 5)

1.558 ± 0.126 (n = 7)

b0.0001

Spinal cord

0.97 ± 0.114 (n = 3)

2.078 ± 0.108 (n = 5)

b0.0001

Bielschowsky

Acute phase (day 18) Transplanted

Control

P value

Transplanted

Control

P value

Corpus callosum Cerebellum Brain stem Spinal cord

0.16 ± 0.03 (n = 5) 0.95 ± 0.088 (n = 5) 1 ± 0.10 (n = 5) 0.81 ± 0.112 (n = 3)

0.662 ± 0.059 (n = 7) 1.218 ± 0.132 (n = 7) 1.804 ± 0.118 (n = 7) 2.035 ± 0.106 (n = 5)

b0.0001 0.093 b0.0001 b0.0001

0.667 ± 0.052 (n = 11) 1.324 ± 0.067 (n = 11) 1.316 ± 0.054 (n = 11) 1.345 ± 0.081 (n = 11)

1.413 ± 0.094 (n = 10) 2.410 ± 0.099 (n = 10) 2.221 ± 0.085 (n = 10) 2.362 ± 0.128 (n = 10)

b0.0001 b0.0001 b0.0001 b0.0001

Kluver Barrera (chronic phase)

Transplanted

Control

P value

Hemispheres (% per section)

0.48 ± 0.38% (n = 7)

2.33 ± 0.59% (n = 9)

0.014

Chronic phase (day 80)

Axonal pathology was reduced in brain of transplanted mice in the acute phase (determined by APP immunohistochemistry and Bielschowsky staining) and in the chronic phase (determined by Bielschowsky staining). The area of demyelination (determined by Kluver Barrera staining) was reduced in the chronic phase of transplanted EAE brains.

may contribute to the inhibitory effect of NPC transplantation in EAE. In addition, the anti-inflammatory effect of NPCs may represent a universal immunosuppressive property of stem cells, similar to that described for embryonic stem cell-derived lines (Fandrich et al., 2002) and bone marrow mesenchymal stem cells (Di Nicola et al., 2002; Djouad et al., 2003; Krampera et al., 2003) which protected them from rejection after transplantation into allogeneic animals. This mechanism may be of major importance in the application of transplantation therapy in immune-mediated diseases, as it could protect the graft from additional immune attack. Thus, while stem cell transplantation in demyelinating diseases was introduced initially as a mean of cell replacement therapy for remyelination, our data suggest that their beneficial effect in EAE may be, in part, via inhibition of brain inflammation leading to reduction in the primary demyelinating process and in the acute axonal injury. In addition, transplanted cells may exert trophic effects on the host brain that enhance host brain cell survival (Ourednik et al., 2002) and endogenous remyelination (Pluchino et al., 2003). Mature astrocytes possess anti-inflammatory properties in vitro (Einstein et al., 2003; Xiao et al., 1998). However, astrocytes did not migrate to the brain after intraventricular transplantation, and did not affect the clinical course of EAE or reduce brain inflammation. We therefore suggest that the clinical and pathological effects of transplanted cells are dependent on their ability to migrate to the active inflammatory-demyelinated lesions. Effective NPC migration enables their close contact with the inflamed tissue, leading to suppression of detrimental inflammation and reduction of demyelination and axonal injury. Thus, the migratory ability of NPC confers an advantage over other cell types in their therapeutic value.

In conclusion, we showed here that neurosphere transplantation attenuates the brain inflammatory process in EAE and reduces the primary processes of demyelination and axonal injury. This is a novel mechanism by which precursor cells convey a beneficial clinical effect in an animal model of multiple sclerosis.

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