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Cytokines production in multiple sclerosis patients after intravenous infusion of autologous mesenchymal stem cells
192
Abstracts
Foundation, Rome, Italy; cDepartment of System Medicine and Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Rome, Italy; dDepartment of Chemistry, Sapienza University, Rome, Italy Introduction: Murine bone marrow mesenchymal stem cells (MSC) can adapt their therapeutic effects to specific microenvironments. In inflammatory conditions, in vitro MSC showed a modulation of genes referable to interferon gamma (IFNg) stimulation, likely involved in the enhancement of the therapeutic plasticity of MSC. The identity of proteins mediating this plasticity is not fully elucidated yet. This study aims at identifying the changes in the protein profile in MSC under IFNg stimulation that can explain the increased neuroprotective and immunomodulatory properties of MSC in inflammatory conditions. Materials and Methods: A Stable Isotope Labeling with Amino acids in cell Culture (SILAC)-based proteomic approach was used. The cells were labeled through the metabolic incorporation of isotopic 13C6Lysine and 13C6-Arginine or 12C6-Lysine and 12C6-Arginine into the proteins expressed by IFNg-primed or unprimed MSC, respectively. Protein identification and quantitation were performed by LC–ESIMS/MS with the Velos Elite Orbitrap mass spectrometer coupled to Mascot Distiller 2.3 interface. The changes in protein expression and signaling pathways represented in the two conditions were analyzed with the Ingenuity Pathways Analysis software. Results: Analysis of the IFNg-primed MSC lysates highlighted an activation of the antigen presentation process, lipid metabolism and response to oxidative stress. Protein synthesis, cell cycle regulation, endoplasmic reticulum stress, along with IFNg-signaling, were also perturbed. IFNg-primed MSC conditioned medium showed an increased level of proteins involved in the regulation of cell–cell and cell–matrix interactions, immunomodulatory and neuroprotective proteins and proteins involved in cholesterol trafficking (analysis in progress). Furthermore, preliminary data suggest that extracellular membrane vesicles released by MSC could shuttle relevant molecules to mediate, at least partially, the immunomodulatory effect of MSC on activated T cells. Discussion and Conclusions: In response to inflammatory stimulation, MSC are able to change profoundly the protein profile and activate specific metabolic and signaling pathways that likely lead to the enhancement of their therapeutic effects. The identification of new regulatory molecules involved in the therapeutic plasticity of MSC paves the way to the study of new therapeutic approaches to treat autoimmune and neurodegenerative diseases. doi:10.1016/j.jneuroim.2014.08.516
262 Influence of experimental autoimmune encephalomyelitis on the localization and viability of neural stem cells after intrathecal transplantation Arianna Merlinia, Donatella De Feoa, Francesca Ruffinia, Elena Brambillaa, Giancarlo Comib, Gianvito Martinoa a
Neuroimmunology Unit, Institute of Experimental Neurology, San Raffaele Scientific Institute, Milan, Italy; bDepartment of Neurology, Institute of Experimental Neurology, San Raffaele Scientific Institute, Milan, Italy Introduction: Intrathecal transplantation of adult neural stem/precursor cells (NPCs) ameliorates disease severity in experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis. However, the kinetics of NPC survival and localization after
intrathecal transplantation and the effect of neuroinflammation on NPC behaviour remain largely unknown. Methods: NPCs were derived from the subventricular zone of 8-week old female C57Bl/6 mice. EAE was induced in syngeneic mice by subcutaneous immunization with myelin oligodendrocyte glycoprotein 35–55 peptide. One million GFP-labelled NPCs were transplanted intrathecally in the cisterna magna of EAE mice at peak of disease severity or healthy matched controls (HC). NPC survival and localization was assessed by immunofluorescence and immunohistochemistry at 1, 7 and 60 days post transplantation (dpt). Each group comprised at least 4 animals. Results: At 1 dpt, NPCs distributed within few millimetres from the injection site (2.46 ± 0.90 mm in EAE; 2.78 ± 0.57 in HC), no further migration was observed at 7 and 60 dpt. At 1 dpt, 8.1% of transplanted NPCs survived in HC and 7.5% in EAE. At 7 dpt the number of surviving NPCs further decreased in both groups (HC: 2.6%; EAE: 4.6%). Indeed, a fraction of transplanted NPCs expressed the apoptotic marker activated caspase 3, with EAE mice showing a trend of reduced apoptosis at 1 dpt (HC: 3.2%; EAE: 1.5%) and at 7 dpt (HC: 3.8%; EAE: 1.4%; p b 0.05). Consistently, at 60 dpt NPCs transplanted in EAE mice displayed increased survival (2.7%) when compared to HC (0.3%; p b 0.05). In both groups, transplanted NPCs localized mainly in the subarachnoid spaces of the fourth ventricle or surrounding meninges at 1 dpt (EAE: 94.8% of surviving NPCs; HC: 87.6%) and 7 dpt (EAE: 98.5%; HC: 89.4%), with a small quota of NPCs integrating in the parenchyma. At 60 dpt, 93.5% of surviving NPCs retained their meningeal localization in the EAE group, while in the HC group 89.4% of the surviving NPCs were found in the parenchyma. Conclusions: The inflammatory environment of EAE does not affect NPC survival in the immediate post-transplant phase. However, 7 and 60 dpt follow-up of transplanted NPCs showed increased survival of NPCs in the EAE group compared to HC as well as different NPC localizations in the two groups, suggesting that neuroinflammation might have long-lasting effects on the behaviour of transplanted NPCs. doi:10.1016/j.jneuroim.2014.08.517
218 Cytokines production in multiple sclerosis patients after intravenous infusion of autologous mesenchymal stem cells Darya Nizheharodava, Mariya Yurkevich, Halina Ivanchyk, Svetlana Kulinich, Marina Zafranskaya Belarussian Medical Academy of Post-Graduate Education, Bio-Medical Research Center, Minsk, Republic Of Belarus Introduction Autoreactive T-lymphocytes and the factors that direct their differentiation into effector phenotypes are the main mediators of multiple sclerosis (MS) pathogenesis. Mesenchymal stem cells (MSC) therapy has the potential to modulate immunological dysregulation of Th1/Th2 cytokine balance associated with MS. Objective:. To estimate the dynamics of cytokines in myelin- and mitogen-induced cell cultures of MS patients before and after MSC administration. Materials and methods: Peripheral blood mononuclear cells (PBMC) were obtained from relapse-remitting MS patients (n = 12) with expanded disability status scale (EDSS) score median of 2,5 before and after intravenous infusion of autologous bone marrow-derived MSC. Extra- and intracellular cytokines profiles were monitored at baseline and 10 days, 1, 3, 6, 9, 12 months after cell therapy with flow cytometer FC500 and ELISA kits. Results:. In vitro study demonstrated the increase of intra- and extracellular interferon gamma (IFN gamma) production in PBMC culture under myelin-stimulated condition (p b 0.05), while the concentrations
Abstracts
of pro- and anti-inflammatory cytokines were elevated in response to mitogen (p b 0.01). The addition of MSC to PBMC culture resulted in significant decrease of IFN gamma intracellular synthesis in both СD4+ and СD8+T-cells as well as extracellular IFN gamma and tumor necrosis factor alpha (TNF alpha) production irrespective of antigenic stimuli (p b 0.01). Therewith, in the presence of MSC in PBMC cultures, the levels of intracellular interleukin-4 (IL-4) myelin-specific production in СD4+Tcells as well as extracellular IL-10 and transforming growth factor beta (TGF beta) secretions were significantly enhanced. In MS patients after MSC administration, the percent of IFN gamma positive T-cells was reduced in both mitogen- and myelin stimulated cell cultures, respectively, after 3 and 6 months of cell therapy (p b 0.05). The number of IL4+T-cells as well as extracellular IL-10 production was increased in response to mitogen as well as myelin antigen, respectively, after 3 and 1 month of MSC administration (p b 0.05). MSC infusion did not significantly affect extracellular TNF alpha and TGF beta dynamic syntheses. Conclusion:. MSC therapy reduced the number of potentially effector T-cells causing demyelination via cytotoxic mechanisms and shifted cytokine balance to immunoregulatory profile promoting the remyelinization in MS patients within at least 6 months after treatment. doi:10.1016/j.jneuroim.2014.08.518
581 Study of phenotypιc changes in cortical neuron cultures induced by mesenchymal stem cells CM during neuroprotection against glutamate-induced cell death Irini Papazian Hellenic Pasteur Institute, Laboratory of Molecular Genetics, Athens, Greece Mesenchymal stem cells (MSCs) are a pluripotent cell subpopulation representing a new therapeutic approach for treating neurodegenerative diseases, such as multiple sclerosis and trauma of central nervous system (CNS). Their therapeutic potential is attributed to neuroprotection and repair of CNS as well as immune regulation although the mechanisms are poorly understood. In this study we investigated short and long-term effects of MSC conditioned medium (MSC CM) in enriched mouse neuronal cortical cultures to gain further insight into the neuroprotective properties of MSCs. Initially, live cell imaging for calcium revealed that the neuroprotection of the MSC CM was associated with reduced glutamate-induced calcium responses in neurons. By using immunocytochemistry we observed that MSC CM did not alter the number or the morphology of neurons, or proportions of GFAP + astrocytes or NG2 + oligodendrocyte precursors in the long-term cultures. We also investigated the differential contribution of soluble and transmembrane TNF in the MSC neuroprotection. Interestingly, the neuroprotective effects of MSC were reduced in neuronal cultures treated with etanercept, a non-selective inhibitor of both forms of TNF but sustained in cultures treated with XPro, a selective inhibitor of soluble TNF. Our results show that the neuroprotective effect of MSC is at least partly mediated via the modulation of glutamate receptors expression and calcium influxing in the neuronal cells, and that transmembrane TNF, most probably expressed by glial cells within the cultures, contributes to neuroprotection. We are currently investigating possible interactions between these two mechanisms. doi:10.1016/j.jneuroim.2014.08.519
193
50 Injection of next-generation directly-induced neural stem cells (iNSCs) induces recovery in a mouse model of multiple sclerosis Luca Peruzzotti-jamettia, Giulia Malluccia, Gillian Tannahilla, Bing Huanga, Yenal Bernard Lakesb, Elena Giustoa, Matteo Donega'a, Beatrice Balzarottia, Frank Edenhoferc, Stefano Pluchinoa a
John van Geest Centre for Brain Repair and Wellcome Trust-Medical Research Council Stem Cell Institute, Department of Clinical Neurosciences, University of Cambridge, Cambridge, United Kingdom; bInstitute of Reconstructive Neurobiology, University of Bonn, Bonn, Germany; c Institute for Anatomy and Cell Biology, University of Würzburg, Würzburg, Germany
Background: Advances in neural stem cell (NSC) biology have raised great expectations that diseases of the CNS may be ameliorated by stem cell therapies. Part drugs and part devices, NSCs work as naturally occurring disease modifying agents that sense signals, migrate to specific sites in the body and execute complex response behaviours. Still, the major limitations of human NSCs therapies are the source from which stem cells are derived (either embryonic or foetal), the immunogenicity of the allogeneic graft, and the genotypic instability over extensive passaging in vitro. The direct conversion of somatic cells (e.g. skin fibroblasts) into induced NSCs (iNSCs) is a promising alternative to conventional stem cell treatments. iNSCs cell reprogramming is direct, highly efficient, and rapid. It requires only one step that is completed within 18 days in vitro, and yields almost 100% colonies of multipotent, stably expandable and autologous NSCs. Objectives: The aim of this work was to assess the therapeutic potential of the intracerebroventricular (icv) transplantation of syngeneic iNSCs in mice affected by chronic experimental autoimmune encephalomyelitis (EAE) as pre-clinical model of multiple sclerosis (MS). Methods: iNSCs from C57BL/6 J somatic skin mouse fibroblasts were grown both as spheres and as adherent monolayers and studied for self-renewal, migratory capacity, multipotency and immune modulation in vitro. We then injected icv 106 GFP + iNSCs, or equal numbers of somatic SVZ-derived GFP + NSCs, in myelin oligodendrocyte glycoprotein (MOG) 35–55 induced EAE mice at disease peak. Behavioural and histopathological outcomes were collected up to 30 days post transplantation. Results: iNSCs showed a significantly high expression of CD44, as well as of the pro-inflammatory chemokine receptors CCR2 and CX3CR1 in vitro. iNSCs also displayed significantly high intrinsic migratory features and anti-inflammatory capacity, when co-cultured (transwell) with LPSactivated macrophages. The icv injection of iNSCs ameliorated chronic EAE in mice. Tissue pathology revealed iNSCs at the level of the injection needle track, close to reactive astrocytes, and within the ventricular system, intermingled with activated microglial cells. Conclusions: While the generation of human iNSCs is envisaged, future experiments will help to understand the role of immunomodulation vs tissue trophism as main mechanisms of iNSC-mediated tissue protection in chronic EAE. doi:10.1016/j.jneuroim.2014.08.520
230 Effects of interferon-beta on neural stem cells and in a mouse model of multiple sclerosis Sarah Stahlkea, Judith Metzdorfa, Xiomara Pedreitturiaa, Simon Van Leeuwenc, Andreas Faissnerc, Ralf Golda, Ingo Kleitera
Abstracts
Foundation, Rome, Italy; cDepartment of System Medicine and Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Rome, Italy; dDepartment of Chemistry, Sapienza University, Rome, Italy Introduction: Murine bone marrow mesenchymal stem cells (MSC) can adapt their therapeutic effects to specific microenvironments. In inflammatory conditions, in vitro MSC showed a modulation of genes referable to interferon gamma (IFNg) stimulation, likely involved in the enhancement of the therapeutic plasticity of MSC. The identity of proteins mediating this plasticity is not fully elucidated yet. This study aims at identifying the changes in the protein profile in MSC under IFNg stimulation that can explain the increased neuroprotective and immunomodulatory properties of MSC in inflammatory conditions. Materials and Methods: A Stable Isotope Labeling with Amino acids in cell Culture (SILAC)-based proteomic approach was used. The cells were labeled through the metabolic incorporation of isotopic 13C6Lysine and 13C6-Arginine or 12C6-Lysine and 12C6-Arginine into the proteins expressed by IFNg-primed or unprimed MSC, respectively. Protein identification and quantitation were performed by LC–ESIMS/MS with the Velos Elite Orbitrap mass spectrometer coupled to Mascot Distiller 2.3 interface. The changes in protein expression and signaling pathways represented in the two conditions were analyzed with the Ingenuity Pathways Analysis software. Results: Analysis of the IFNg-primed MSC lysates highlighted an activation of the antigen presentation process, lipid metabolism and response to oxidative stress. Protein synthesis, cell cycle regulation, endoplasmic reticulum stress, along with IFNg-signaling, were also perturbed. IFNg-primed MSC conditioned medium showed an increased level of proteins involved in the regulation of cell–cell and cell–matrix interactions, immunomodulatory and neuroprotective proteins and proteins involved in cholesterol trafficking (analysis in progress). Furthermore, preliminary data suggest that extracellular membrane vesicles released by MSC could shuttle relevant molecules to mediate, at least partially, the immunomodulatory effect of MSC on activated T cells. Discussion and Conclusions: In response to inflammatory stimulation, MSC are able to change profoundly the protein profile and activate specific metabolic and signaling pathways that likely lead to the enhancement of their therapeutic effects. The identification of new regulatory molecules involved in the therapeutic plasticity of MSC paves the way to the study of new therapeutic approaches to treat autoimmune and neurodegenerative diseases. doi:10.1016/j.jneuroim.2014.08.516
262 Influence of experimental autoimmune encephalomyelitis on the localization and viability of neural stem cells after intrathecal transplantation Arianna Merlinia, Donatella De Feoa, Francesca Ruffinia, Elena Brambillaa, Giancarlo Comib, Gianvito Martinoa a
Neuroimmunology Unit, Institute of Experimental Neurology, San Raffaele Scientific Institute, Milan, Italy; bDepartment of Neurology, Institute of Experimental Neurology, San Raffaele Scientific Institute, Milan, Italy Introduction: Intrathecal transplantation of adult neural stem/precursor cells (NPCs) ameliorates disease severity in experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis. However, the kinetics of NPC survival and localization after
intrathecal transplantation and the effect of neuroinflammation on NPC behaviour remain largely unknown. Methods: NPCs were derived from the subventricular zone of 8-week old female C57Bl/6 mice. EAE was induced in syngeneic mice by subcutaneous immunization with myelin oligodendrocyte glycoprotein 35–55 peptide. One million GFP-labelled NPCs were transplanted intrathecally in the cisterna magna of EAE mice at peak of disease severity or healthy matched controls (HC). NPC survival and localization was assessed by immunofluorescence and immunohistochemistry at 1, 7 and 60 days post transplantation (dpt). Each group comprised at least 4 animals. Results: At 1 dpt, NPCs distributed within few millimetres from the injection site (2.46 ± 0.90 mm in EAE; 2.78 ± 0.57 in HC), no further migration was observed at 7 and 60 dpt. At 1 dpt, 8.1% of transplanted NPCs survived in HC and 7.5% in EAE. At 7 dpt the number of surviving NPCs further decreased in both groups (HC: 2.6%; EAE: 4.6%). Indeed, a fraction of transplanted NPCs expressed the apoptotic marker activated caspase 3, with EAE mice showing a trend of reduced apoptosis at 1 dpt (HC: 3.2%; EAE: 1.5%) and at 7 dpt (HC: 3.8%; EAE: 1.4%; p b 0.05). Consistently, at 60 dpt NPCs transplanted in EAE mice displayed increased survival (2.7%) when compared to HC (0.3%; p b 0.05). In both groups, transplanted NPCs localized mainly in the subarachnoid spaces of the fourth ventricle or surrounding meninges at 1 dpt (EAE: 94.8% of surviving NPCs; HC: 87.6%) and 7 dpt (EAE: 98.5%; HC: 89.4%), with a small quota of NPCs integrating in the parenchyma. At 60 dpt, 93.5% of surviving NPCs retained their meningeal localization in the EAE group, while in the HC group 89.4% of the surviving NPCs were found in the parenchyma. Conclusions: The inflammatory environment of EAE does not affect NPC survival in the immediate post-transplant phase. However, 7 and 60 dpt follow-up of transplanted NPCs showed increased survival of NPCs in the EAE group compared to HC as well as different NPC localizations in the two groups, suggesting that neuroinflammation might have long-lasting effects on the behaviour of transplanted NPCs. doi:10.1016/j.jneuroim.2014.08.517
218 Cytokines production in multiple sclerosis patients after intravenous infusion of autologous mesenchymal stem cells Darya Nizheharodava, Mariya Yurkevich, Halina Ivanchyk, Svetlana Kulinich, Marina Zafranskaya Belarussian Medical Academy of Post-Graduate Education, Bio-Medical Research Center, Minsk, Republic Of Belarus Introduction Autoreactive T-lymphocytes and the factors that direct their differentiation into effector phenotypes are the main mediators of multiple sclerosis (MS) pathogenesis. Mesenchymal stem cells (MSC) therapy has the potential to modulate immunological dysregulation of Th1/Th2 cytokine balance associated with MS. Objective:. To estimate the dynamics of cytokines in myelin- and mitogen-induced cell cultures of MS patients before and after MSC administration. Materials and methods: Peripheral blood mononuclear cells (PBMC) were obtained from relapse-remitting MS patients (n = 12) with expanded disability status scale (EDSS) score median of 2,5 before and after intravenous infusion of autologous bone marrow-derived MSC. Extra- and intracellular cytokines profiles were monitored at baseline and 10 days, 1, 3, 6, 9, 12 months after cell therapy with flow cytometer FC500 and ELISA kits. Results:. In vitro study demonstrated the increase of intra- and extracellular interferon gamma (IFN gamma) production in PBMC culture under myelin-stimulated condition (p b 0.05), while the concentrations
Abstracts
of pro- and anti-inflammatory cytokines were elevated in response to mitogen (p b 0.01). The addition of MSC to PBMC culture resulted in significant decrease of IFN gamma intracellular synthesis in both СD4+ and СD8+T-cells as well as extracellular IFN gamma and tumor necrosis factor alpha (TNF alpha) production irrespective of antigenic stimuli (p b 0.01). Therewith, in the presence of MSC in PBMC cultures, the levels of intracellular interleukin-4 (IL-4) myelin-specific production in СD4+Tcells as well as extracellular IL-10 and transforming growth factor beta (TGF beta) secretions were significantly enhanced. In MS patients after MSC administration, the percent of IFN gamma positive T-cells was reduced in both mitogen- and myelin stimulated cell cultures, respectively, after 3 and 6 months of cell therapy (p b 0.05). The number of IL4+T-cells as well as extracellular IL-10 production was increased in response to mitogen as well as myelin antigen, respectively, after 3 and 1 month of MSC administration (p b 0.05). MSC infusion did not significantly affect extracellular TNF alpha and TGF beta dynamic syntheses. Conclusion:. MSC therapy reduced the number of potentially effector T-cells causing demyelination via cytotoxic mechanisms and shifted cytokine balance to immunoregulatory profile promoting the remyelinization in MS patients within at least 6 months after treatment. doi:10.1016/j.jneuroim.2014.08.518
581 Study of phenotypιc changes in cortical neuron cultures induced by mesenchymal stem cells CM during neuroprotection against glutamate-induced cell death Irini Papazian Hellenic Pasteur Institute, Laboratory of Molecular Genetics, Athens, Greece Mesenchymal stem cells (MSCs) are a pluripotent cell subpopulation representing a new therapeutic approach for treating neurodegenerative diseases, such as multiple sclerosis and trauma of central nervous system (CNS). Their therapeutic potential is attributed to neuroprotection and repair of CNS as well as immune regulation although the mechanisms are poorly understood. In this study we investigated short and long-term effects of MSC conditioned medium (MSC CM) in enriched mouse neuronal cortical cultures to gain further insight into the neuroprotective properties of MSCs. Initially, live cell imaging for calcium revealed that the neuroprotection of the MSC CM was associated with reduced glutamate-induced calcium responses in neurons. By using immunocytochemistry we observed that MSC CM did not alter the number or the morphology of neurons, or proportions of GFAP + astrocytes or NG2 + oligodendrocyte precursors in the long-term cultures. We also investigated the differential contribution of soluble and transmembrane TNF in the MSC neuroprotection. Interestingly, the neuroprotective effects of MSC were reduced in neuronal cultures treated with etanercept, a non-selective inhibitor of both forms of TNF but sustained in cultures treated with XPro, a selective inhibitor of soluble TNF. Our results show that the neuroprotective effect of MSC is at least partly mediated via the modulation of glutamate receptors expression and calcium influxing in the neuronal cells, and that transmembrane TNF, most probably expressed by glial cells within the cultures, contributes to neuroprotection. We are currently investigating possible interactions between these two mechanisms. doi:10.1016/j.jneuroim.2014.08.519
193
50 Injection of next-generation directly-induced neural stem cells (iNSCs) induces recovery in a mouse model of multiple sclerosis Luca Peruzzotti-jamettia, Giulia Malluccia, Gillian Tannahilla, Bing Huanga, Yenal Bernard Lakesb, Elena Giustoa, Matteo Donega'a, Beatrice Balzarottia, Frank Edenhoferc, Stefano Pluchinoa a
John van Geest Centre for Brain Repair and Wellcome Trust-Medical Research Council Stem Cell Institute, Department of Clinical Neurosciences, University of Cambridge, Cambridge, United Kingdom; bInstitute of Reconstructive Neurobiology, University of Bonn, Bonn, Germany; c Institute for Anatomy and Cell Biology, University of Würzburg, Würzburg, Germany
Background: Advances in neural stem cell (NSC) biology have raised great expectations that diseases of the CNS may be ameliorated by stem cell therapies. Part drugs and part devices, NSCs work as naturally occurring disease modifying agents that sense signals, migrate to specific sites in the body and execute complex response behaviours. Still, the major limitations of human NSCs therapies are the source from which stem cells are derived (either embryonic or foetal), the immunogenicity of the allogeneic graft, and the genotypic instability over extensive passaging in vitro. The direct conversion of somatic cells (e.g. skin fibroblasts) into induced NSCs (iNSCs) is a promising alternative to conventional stem cell treatments. iNSCs cell reprogramming is direct, highly efficient, and rapid. It requires only one step that is completed within 18 days in vitro, and yields almost 100% colonies of multipotent, stably expandable and autologous NSCs. Objectives: The aim of this work was to assess the therapeutic potential of the intracerebroventricular (icv) transplantation of syngeneic iNSCs in mice affected by chronic experimental autoimmune encephalomyelitis (EAE) as pre-clinical model of multiple sclerosis (MS). Methods: iNSCs from C57BL/6 J somatic skin mouse fibroblasts were grown both as spheres and as adherent monolayers and studied for self-renewal, migratory capacity, multipotency and immune modulation in vitro. We then injected icv 106 GFP + iNSCs, or equal numbers of somatic SVZ-derived GFP + NSCs, in myelin oligodendrocyte glycoprotein (MOG) 35–55 induced EAE mice at disease peak. Behavioural and histopathological outcomes were collected up to 30 days post transplantation. Results: iNSCs showed a significantly high expression of CD44, as well as of the pro-inflammatory chemokine receptors CCR2 and CX3CR1 in vitro. iNSCs also displayed significantly high intrinsic migratory features and anti-inflammatory capacity, when co-cultured (transwell) with LPSactivated macrophages. The icv injection of iNSCs ameliorated chronic EAE in mice. Tissue pathology revealed iNSCs at the level of the injection needle track, close to reactive astrocytes, and within the ventricular system, intermingled with activated microglial cells. Conclusions: While the generation of human iNSCs is envisaged, future experiments will help to understand the role of immunomodulation vs tissue trophism as main mechanisms of iNSC-mediated tissue protection in chronic EAE. doi:10.1016/j.jneuroim.2014.08.520
230 Effects of interferon-beta on neural stem cells and in a mouse model of multiple sclerosis Sarah Stahlkea, Judith Metzdorfa, Xiomara Pedreitturiaa, Simon Van Leeuwenc, Andreas Faissnerc, Ralf Golda, Ingo Kleitera