RESUMO
Transplantation of mesenchymal stem cells (MSCs) into injured or diseased tissue-for the in situ delivery of a wide variety of MSC-secreted therapeutic proteins-is an emerging approach for the modulation of the clinical course of several diseases and traumata. From an emergency point-of-view, allogeneic MSCs have numerous advantages over patient-specific autologous MSCs since "off-the-shelf" cell preparations could be readily available for instant therapeutic intervention following acute injury. Although we confirmed the in vitro immunomodulatory capacity of allogeneic MSCs on antigen-presenting cells with standard coculture experiments, allogeneic MSC grafts were irrevocably rejected by the host's immune system upon either intramuscular or intracerebral transplantation. In an attempt to modulate MSC allograft rejection in vivo, we transduced MSCs with an interleukin-13 (IL13)-expressing lentiviral vector. Our data clearly indicate that prolonged survival of IL13-expressing allogeneic MSC grafts in muscle tissue coincided with the induction of an alternatively activated macrophage phenotype in vivo and a reduced number of alloantigen-reactive IFNγ- and/or IL2-producing CD8(+) T cells compared to nonmodified allografts. Similarly, intracerebral IL13-expressing MSC allografts also exhibited prolonged survival and induction of an alternatively activated macrophage phenotype, although a peripheral T cell component was absent. In summary, this study demonstrates that both innate and adaptive immune responses are effectively modulated in vivo by locally secreted IL13, ultimately resulting in prolonged MSC allograft survival in both muscle and brain tissue. Stem Cells 2016;34:1971-1984.
Assuntos
Sobrevivência de Enxerto/imunologia , Interleucina-13/farmacologia , Isoantígenos/imunologia , Ativação Linfocitária/efeitos dos fármacos , Macrófagos/metabolismo , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Linfócitos T/imunologia , Aloenxertos/efeitos dos fármacos , Aloenxertos/imunologia , Animais , Formação de Anticorpos/efeitos dos fármacos , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Engenharia Genética , Imunomodulação/efeitos dos fármacos , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Camundongos , Microglia/efeitos dos fármacos , Microglia/patologia , Linfócitos T/efeitos dos fármacosRESUMO
Detrimental inflammatory responses in the central nervous system are a hallmark of various brain injuries and diseases. With this study we provide evidence that lentiviral vector-mediated expression of the immune-modulating cytokine interleukin 13 (IL-13) induces an alternative activation program in both microglia and macrophages conferring protection against severe oligodendrocyte loss and demyelination in the cuprizone mouse model for multiple sclerosis (MS). First, IL-13 mediated modulation of cuprizone induced lesions was monitored using T2 -weighted magnetic resonance imaging and magnetization transfer imaging, and further correlated with quantitative histological analyses for inflammatory cell influx, oligodendrocyte death, and demyelination. Second, following IL-13 immune gene therapy in cuprizone-treated eGFP+ bone marrow chimeric mice, we provide evidence that IL-13 directs the polarization of both brain-resident microglia and infiltrating macrophages towards an alternatively activated phenotype, thereby promoting the conversion of a pro-inflammatory environment toward an anti-inflammatory environment, as further evidenced by gene expression analyses. Finally, we show that IL-13 immune gene therapy is also able to limit lesion severity in a pre-existing inflammatory environment. In conclusion, these results highlight the potential of IL-13 to modulate microglia/macrophage responses and to improve disease outcome in a mouse model for MS. GLIA 2016;64:2181-2200.
Assuntos
Doenças Desmielinizantes/terapia , Encefalite/terapia , Terapia Genética/métodos , Interleucina-13 , Macrófagos/efeitos dos fármacos , Microglia/efeitos dos fármacos , Animais , Antígenos de Diferenciação/metabolismo , Transplante de Medula Óssea , Cuprizona/toxicidade , Citocinas/genética , Citocinas/metabolismo , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/diagnóstico por imagem , Modelos Animais de Doenças , Encefalite/induzido quimicamente , Encefalite/diagnóstico por imagem , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Interleucina-13/genética , Interleucina-13/metabolismo , Interleucina-13/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Inibidores da Monoaminoxidase/toxicidade , Proteínas da Mielina/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Transdução GenéticaRESUMO
BACKGROUND: Promoting the neuroprotective and repair-inducing effector functions of microglia and macrophages, by means of M2 polarisation or alternative activation, is expected to become a new therapeutic approach for central nervous system (CNS) disorders in which detrimental pro-inflammatory microglia and/or macrophages display a major contribution to the neuropathology. In this study, we present a novel in vivo approach using intracerebral grafting of mesenchymal stem cells (MSC) genetically engineered to secrete interleukin 13 (IL13-MSC). METHODS: In the first experimental setup, control MSC and IL13-MSC were grafted in the CNS of eGFP+ bone marrow chimaeric C57BL/6 mice to histologically evaluate IL13-mediated expression of several markers associated with alternative activation, including arginase1 and Ym1, on MSC graft-recognising microglia and MSC graft-infiltrating macrophages. In the second experimental setup, IL13-MSC were grafted on the right side (or on both the right and left sides) of the splenium of the corpus callosum in wild-type C57BL/6 mice and in C57BL/6 CX3CR1eGFP/+CCR2RFP/+ transgenic mice. Next, CNS inflammation and demyelination was induced by means of a cuprizone-supplemented diet. The influence of IL13-MSC grafting on neuropathological alterations was monitored by non-invasive T 2-weighted magnetic resonance imaging (MRI) and quantitative histological analyses, as compared to cuprizone-treated mice with control MSC grafts and/or cuprizone-treated mice without MSC injection. RESULTS: In the first part of this study, we demonstrate that MSC graft-associated microglia and MSC graft-infiltrating macrophages are forced into alternative activation upon grafting of IL13-MSC, but not upon grafting of control MSC. In the second part of this study, we demonstrate that grafting of IL13-MSC, in addition to the recruitment of M2 polarised macrophages, limits cuprizone-induced microgliosis, oligodendrocyte death and demyelination. Furthermore, we here demonstrate that injection of IL13-MSC at both sides of the splenium leads to a superior protective effect as compared to a single injection at one side of the splenium. CONCLUSIONS: Controlled and localised production of IL13 by means of intracerebral MSC grafting has the potential to modulate cell graft- and pathology-associated microglial/macrophage responses, and to interfere with oligodendrocyte death and demyelinating events in the cuprizone mouse model.
Assuntos
Cuprizona/toxicidade , Doenças Desmielinizantes , Gliose/etiologia , Interleucina-13/metabolismo , Transplante de Células-Tronco Mesenquimais , Inibidores da Monoaminoxidase/toxicidade , Oligodendroglia/patologia , Animais , Linhagem Celular Transformada , Citocinas/genética , Citocinas/metabolismo , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/diagnóstico por imagem , Doenças Desmielinizantes/patologia , Doenças Desmielinizantes/cirurgia , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imageamento por Ressonância Magnética , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Básica da Mielina/metabolismo , Oligodendroglia/efeitos dos fármacosRESUMO
Multiple sclerosis is a devastating demyelinating disease of the central nervous system (CNS) in which endogenous remyelination, and thus recovery, often fails. Although the cuprizone mouse model allowed elucidation of many molecular factors governing remyelination, currently very little is known about the spatial origin of the oligodendrocyte progenitor cells that initiate remyelination in this model. Therefore, we here investigated in this model whether subventricular zone (SVZ) neural stem/progenitor cells (NSPCs) contribute to remyelination of the splenium following cuprizone-induced demyelination. Experimentally, from the day of in situ NSPC labeling, C57BL/6J mice were fed a 0.2% cuprizone diet during a 4-week period and then left to recover on a normal diet for 8weeks. Two in situ labeling strategies were employed: (i) NSPCs were labeled by intraventricular injection of micron-sized iron oxide particles and then followed up longitudinally by means of magnetic resonance imaging (MRI), and (ii) SVZ NSPCs were transduced with a lentiviral vector encoding the eGFP and Luciferase reporter proteins for longitudinal monitoring by means of in vivo bioluminescence imaging (BLI). In contrast to preceding suggestions, no migration of SVZ NSPC towards the demyelinated splenium was observed using both MRI and BLI, and further validated by histological analysis, thereby demonstrating that SVZ NSPCs are unable to contribute directly to remyelination of the splenium in the cuprizone model. Interestingly, using longitudinal BLI analysis and confirmed by histological analysis, an increased migration of SVZ NSPC-derived neuroblasts towards the olfactory bulb was observed following cuprizone treatment, indicative for a potential link between CNS inflammation and increased neurogenesis.
Assuntos
Ventrículos Cerebrais/patologia , Corpo Caloso/patologia , Doenças Desmielinizantes/patologia , Imagem de Difusão por Ressonância Magnética/métodos , Fibras Nervosas Mielinizadas/patologia , Células-Tronco Neurais/patologia , Bulbo Olfatório/patologia , Animais , Movimento Celular , Rastreamento de Células/métodos , Cuprizona , Doenças Desmielinizantes/induzido quimicamente , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência/métodos , Imagem Multimodal/métodos , Vias Neurais/patologia , NeurogêneseRESUMO
BACKGROUND: Despite positive reports on the efficacy of stem cell therapy for the treatment of cardiovascular disease, the nature of stem cell homing to ischemic tissues remains elusive. RESULTS: We used a mouse model of peripheral tissue ischemia to study the survival and homing capacity of dual reporter gene (eGFP/Luciferase) expressing bone marrow-derived stromal cells (BMSC). Cell homing and survival were studied in the presence and absence of ciclosporin A (CsA) immunosuppression using bioluminescence imaging (BLI) together with confocal endomicroscopy. Different injection strategies were applied: central venous (CV), intra-arterial (IA) and intramuscular (IM). BLI and confocal endomicroscopy evidenced complete rejection of the IM injected allogeneic BMSC transplant within 5 to 10 days. Immunosuppression with CsA could only marginally prolong graft survival. IM injected BMSC did not migrate to the site of the arterial ligation. CV injection of BMSC resulted in massive pulmonary infarction, leading to respiratory failure and death. Intrapulmonary cell trapping was evidenced by confocal endomicroscopy, BLI and fluorescence microscopy. IA injection of BMSC proved to be a feasible and safe strategy to bypass the lung circulation. During the follow-up period, neither BLI nor confocal endomicroscopy revealed any convincing ischemia-directed homing of BMSC. CONCLUSIONS: BLI and confocal endomicroscopy are complementary imaging techniques for studying the in vivo biology of dual reporter gene-expressing BMSC. Allogeneic BMSC survival is limited in an immunocompetent host and cannot be preserved by CsA immunosuppression alone. We did not find substantial evidence for ischemia-directed BMSC homing and caution against CV injection of BMSC, which can lead to massive pulmonary infarction.
Assuntos
Células-Tronco Mesenquimais/citologia , Animais , Ciclosporina/farmacologia , Modelos Animais de Doenças , Genes Reporter , Sobrevivência de Enxerto/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Isquemia/terapia , Luciferases/genética , Luciferases/metabolismo , Medições Luminescentes , Masculino , Transplante de Células-Tronco Mesenquimais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Transplante HomólogoRESUMO
Currently, much attention is given to the development of cellular therapies for treatment of central nervous system (CNS) injuries. Diverse cell implantation strategies, either to directly replace damaged neural tissue or to create a neuroregenerative environment, are proposed to restore impaired brain function. However, because of the complexity of the CNS, it is now becoming clear that the contribution of cell implantation into the brain will mainly act in a supportive manner. In addition, given the time dependence of neural development during embryonic and post-natal life, cellular implants, either self or non-self, will most likely have to interact for a sustained period of time with both healthy and injured neural tissue. The latter also implies potential recognition of cellular implants by the innate immune system of the brain. In this review, we will emphasize on preclinical observations in rodents, regarding the recognition and immunogenicity of autologous, allogeneic and xenogeneic cellular implants in the CNS of immune-competent hosts. Taken together, we here suggest that a profound study of the interaction between cellular grafts and the brain's innate immune system will be inevitable before clinical cell transplantation in the CNS can be performed successfully.
Assuntos
Encéfalo/imunologia , Transplante de Células , Imunidade Inata/imunologia , Imunologia de Transplantes/imunologia , Animais , Terapia Baseada em Transplante de Células e Tecidos , Doenças do Sistema Nervoso Central/terapia , HumanosRESUMO
BACKGROUND: Cell transplantation is likely to become an important therapeutic tool for the treatment of various traumatic and ischemic injuries to the central nervous system (CNS). However, in many pre-clinical cell therapy studies, reporter gene-assisted imaging of cellular implants in the CNS and potential reporter gene and/or cell-based immunogenicity, still remain challenging research topics. RESULTS: In this study, we performed cell implantation experiments in the CNS of immunocompetent mice using autologous (syngeneic) luciferase-expressing bone marrow-derived stromal cells (BMSC-Luc) cultured from ROSA26-L-S-L-Luciferase transgenic mice, and BMSC-Luc genetically modified using a lentivirus encoding the enhanced green fluorescence protein (eGFP) and the puromycin resistance gene (Pac) (BMSC-Luc/eGFP/Pac). Both reporter gene-modified BMSC populations displayed high engraftment capacity in the CNS of immunocompetent mice, despite potential immunogenicity of introduced reporter proteins, as demonstrated by real-time bioluminescence imaging (BLI) and histological analysis at different time-points post-implantation. In contrast, both BMSC-Luc and BMSC-Luc/eGFP/Pac did not survive upon intramuscular cell implantation, as demonstrated by real-time BLI at different time-points post-implantation. In addition, ELISPOT analysis demonstrated the induction of IFN-gamma-producing CD8+ T-cells upon intramuscular cell implantation, but not upon intracerebral cell implantation, indicating that BMSC-Luc and BMSC-Luc/eGFP/Pac are immune-tolerated in the CNS. However, in our experimental transplantation model, results also indicated that reporter gene-specific immune-reactive T-cell responses were not the main contributors to the immunological rejection of BMSC-Luc or BMSC-Luc/eGFP/Pac upon intramuscular cell implantation. CONCLUSION: We here demonstrate that reporter gene-modified BMSC derived from ROSA26-L-S-L-Luciferase transgenic mice are immune-tolerated upon implantation in the CNS of syngeneic immunocompetent mice, providing a research model for studying survival and localisation of autologous BMSC implants in the CNS by real-time BLI and/or histological analysis in the absence of immunosuppressive therapy.
Assuntos
Células da Medula Óssea/citologia , Encéfalo , Luciferases/genética , Células Estromais/transplante , Tolerância ao Transplante , Animais , Encéfalo/metabolismo , Células Cultivadas , Diagnóstico por Imagem , Genes Reporter , Luciferases/metabolismo , Substâncias Luminescentes/metabolismo , Medições Luminescentes , Masculino , Camundongos , Camundongos Transgênicos , Modelos AnimaisRESUMO
Although intracerebral transplantation of various fibroblast(-like) cell populations has been shown feasible, little is known about the actual in vivo remodeling of these cellular grafts and their environment. In this study, we aimed to compare the in vitro and in vivo behavior of two phenotypically similar-but developmentally distinct-fibroblast-like cell populations, namely, mouse embryonic fibroblasts (mEFs) and mouse fetal membrane-derived stromal cells (mFMSCs). While both mEFs and mFMSCs are readily able to reduce TNF-α secretion by LPS/IFN-γ-activated BV-2 microglia, mFMSCs and mEFs display strikingly opposite behavior with regard to VEGF production under normal and inflammatory conditions. Whereas mFMSCs downregulate VEGF production upon coculture with LPS/IFN-γ-activated BV-2 microglia, mEFs upregulate VEGF production in the presence of LPS/IFN-γ-activated BV-2 microglia. Subsequently, in vivo grafting of mFMSCs and mEFs revealed no difference in microglial and astroglial responses toward the cellular grafts. However, mFMSC grafts displayed a lower degree of neoangiogenesis compared to mEF grafts, thereby potentially explaining the lower cell number able to survive in mFMSC grafts. In summary, our results suggest that physiological differences between fibroblast-like cell populations might lie at the basis of variations in histopathological and/or clinical outcome following cell grafting in mouse brain.
Assuntos
Encéfalo/patologia , Embrião de Mamíferos/citologia , Membranas Extraembrionárias/citologia , Fibroblastos/transplante , Células Estromais/transplante , Animais , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Imunofenotipagem , Interferon gama/farmacologia , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Células Estromais/citologia , Células Estromais/metabolismo , Transplante Homólogo , Fator de Necrose Tumoral alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
In this study, the interaction between cells and micron-sized paramagnetic iron oxide (MPIO) particles was investigated by characterizing MPIO in their original state, and after cellular uptake in vitro as well as in vivo. Moreover, MPIO in the olfactory bulb were studied 9 months after injection. Using various imaging techniques, cell-MPIO interactions were investigated with increasing spatial resolution. Live cell confocal microscopy demonstrated that MPIO co-localize with lysosomes after in vitro cellular uptake. In more detail, a membrane surrounding the MPIO was observed by high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM). Following MPIO uptake in vivo, the same cell-MPIO interaction was observed by HAADF-STEM in the subventricular zone at 1 week and in the olfactory bulb at 9 months after MPIO injection. These findings provide proof for the current hypothesis that MPIO are internalized by the cell through endocytosis. The results also show MPIO are not biodegradable, even after 9 months in the brain. Moreover, they show the possibility of HAADF-STEM generating information on the labeled cell as well as on the MPIO. In summary, the methodology presented here provides a systematic route to investigate the interaction between cells and nanoparticles from the micrometer level down to the nanometer level and beyond.
Assuntos
Compostos Férricos , Células-Tronco Mesenquimais/ultraestrutura , Imagem Multimodal/métodos , Bulbo Olfatório/ultraestrutura , Animais , Linhagem Celular , Rastreamento de Células/métodos , Endocitose/efeitos dos fármacos , Compostos Férricos/química , Humanos , Lisossomos/química , Nanopartículas Metálicas/química , Camundongos , Microscopia Eletrônica de Transmissão e Varredura , Tamanho da Partícula , Coloração e RotulagemRESUMO
Preclinical animal studies involving intracerebral (stem) cell grafting are gaining popularity in many laboratories due to the reported beneficial effects of cell grafting on various diseases or traumata of the central nervous system (CNS). In this chapter, we describe a histological workflow to characterize and quantify cellular events following neural and fibroblast(-like) stem cell grafting in healthy and demyelinated CNS tissue. First, we provide standardized protocols to isolate and culture eGFP(+) neural and fibroblast(-like) stem cells from embryonic mouse tissue. Second, we describe flow cytometric procedures to determine cell viability, eGFP transgene expression, and the expression of different stem cell lineage markers. Third, we explain how to induce reproducible demyelination in the CNS of mice by means of cuprizone administration, a validated mouse model for human multiple sclerosis. Fourth, the technical procedures for cell grafting in the CNS are explained in detail. Finally, an optimized and validated workflow for the quantitative histological analysis of cell graft survival and endogenous astroglial and microglial responses is provided.
Assuntos
Doenças Desmielinizantes/terapia , Células-Tronco Neurais/citologia , Transplante de Células-Tronco , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Terapia Baseada em Transplante de Células e Tecidos , Modelos Animais de Doenças , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Citometria de Fluxo , Expressão Gênica , Genes Reporter , Sobrevivência de Enxerto , Imuno-Histoquímica , Camundongos , Células-Tronco Neurais/metabolismo , Medicina Regenerativa , TransgenesRESUMO
Over the past decade a lot of research has been performed towards the therapeutic use of mesenchymal stem cells (MSCs) in neurodegenerative and neuroinflammatory diseases. MSCs have shown to be beneficial in different preclinical studies of central nervous system (CNS) disorders due to their immunomodulatory properties and their capacity to secrete various growth factors. Nevertheless, most of the transplanted cells die within the first hours after transplantation and induce a neuroinflammatory response. In order to increase the efficacy of MSC transplantation, it is thus imperative to completely characterise the mechanisms mediating neuroinflammation and cell death following MSC transplantation into the CNS. Consequently, different components of these cell death- and neuroinflammation-inducing pathways can be targeted in an attempt to improve the therapeutic potential of MSCs for CNS disorders.
Assuntos
Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/fisiologia , Sistema Nervoso Central , Humanos , Células-Tronco Mesenquimais/patologiaRESUMO
Stem cell transplantation in the central nervous system (CNS) is currently under intensive investigation as a novel therapeutic approach for a variety of brain disorders and/or injuries. However, one of the main hurdles at the moment is the lack of standardized procedures to evaluate cell graft survival and behavior following transplantation into CNS tissue, thereby leading to the publication of confusing and/or conflicting research results. In this chapter, we therefore provide validated in vivo bioluminescence and postmortem histological procedures to quantitatively determine: (a) the survival of grafted stem cells, and (b) the microglial and astroglial cell responses following cell grafting.
Assuntos
Sistema Nervoso Central/citologia , Medições Luminescentes/métodos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Neurais/transplante , Animais , Astrócitos/citologia , Diferenciação Celular , Sobrevivência Celular , Terapia Baseada em Transplante de Células e Tecidos , Células Cultivadas , Sobrevivência de Enxerto , Células-Tronco Mesenquimais/citologia , Camundongos , Microglia/citologia , Células-Tronco Neurais/citologia , Neurônios/metabolismo , Oligodendroglia/metabolismoRESUMO
Although cell transplantation is increasingly suggested to be beneficial for the treatment of various neurodegenerative diseases, the therapeutic application of such intervention is currently hindered by the limited knowledge regarding central nervous system (CNS) transplantation immunology. In this study, we aimed to investigate the early post transplantation innate immune events following grafting of autologous mesenchymal stromal cells (MSC) in the CNS of immune competent mice. First, the survival of grafted Luciferase/eGFP-expressing MSC (MSC-Luc/eGFP) was demonstrated to be stable from on day 3 post implantation using in vivo bioluminescence imaging (BLI), which was further confirmed by quantitative histological analysis of MSC-Luc/eGFP graft survival. Additional histological analyses at week 1 and week 2 post grafting revealed the appearance of (i) graft-surrounding/-invading Iba1+ microglia and (ii) graft-surrounding GFAP+ astrocytes, as compared to day 0 post grafting. While the density of graft-surrounding astrocytes and microglia did not change between week 1 and week 2 post grafting, the density of graft-invading microglia significantly decreased between week 1 and week 2 post implantation. However, despite the observed decrease in microglial density within the graft site, additional phenotypic analysis of graft-invading microglia, based on CD11b- and MHCII-expression, revealed >50% of graft-invading microglia at week 2 post implantation to display an activated status. Although microglial expression of CD11b and MHCII is already suggestive for a pro-inflammatory M1-oriented phenotype, the latter was further confirmed by: (i) the expression of NOS2 by microglia within the graft site, and (ii) the absence of arginase 1-expression, an enzyme known to suppress NO activity in M2-oriented microglia, on graft-surrounding and -invading microglia. In summary, we here provide a detailed phenotypic analysis of post transplantation innate immune events in the CNS of mice, and warrant that such intervention is associated with an M1-oriented microglia response and severe astrogliosis.
Assuntos
Astrócitos/citologia , Encéfalo/citologia , Sobrevivência de Enxerto/imunologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Microglia/citologia , Animais , Astrócitos/imunologia , Encéfalo/imunologia , Antígeno CD11b/genética , Antígeno CD11b/imunologia , Contagem de Células , Células Cultivadas , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde , Luciferases , Masculino , Células-Tronco Mesenquimais/imunologia , Camundongos , Camundongos Transgênicos , Microglia/imunologia , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/imunologia , Transplante AutólogoRESUMO
Stem cell transplantation holds great promise for restoration of neural function in various neurodegenerative disorders, including multiple sclerosis (MS). However, many questions remain regarding the true efficacy and precise mode of action of stem cell-based therapeutic approaches. Therefore, in this article, we will first discuss the ideal route and/or timing of stem cell-based therapies for experimental autoimmune encephalomyelitis (EAE), the most used preclinical animal model for MS. Next, we will provide an overview of the proposed mechanisms that contribute to the beneficial effects of stem cell transplantation observed during the treatment of rodent EAE. Reviews of current and past literature clearly demonstrate conceptual changes in the development of stem cell-based approaches for EAE/MS, leading to the identification of several major challenges to be tackled before (stem) cell therapy for rodent EAE can be safely and successfully translated to human therapy for MS.
Assuntos
Esclerose Múltipla/terapia , Transplante de Células-Tronco , Células-Tronco/citologia , Animais , Diferenciação Celular , Movimento Celular , Humanos , Inflamação/patologiaRESUMO
INTRODUCTION: Transplantation of neural stem cells (NSCs) is increasingly suggested to become part of future therapeutic approaches to improve functional outcome of various central nervous system disorders. However, recently it has become clear that only a small fraction of grafted NSCs display long-term survival in the (injured) adult mouse brain. Given the clinical invasiveness of NSC grafting into brain tissue, profound characterisation and understanding of early post-transplantation events is imperative to claim safety and efficacy of cell-based interventions. METHODS: Here, we applied in vivo bioluminescence imaging (BLI) and post-mortem quantitative histological analysis to determine the localisation and survival of grafted NSCs at early time points post-transplantation. RESULTS: An initial dramatic cell loss (up to 80% of grafted cells) due to apoptosis could be observed within the first 24 hours post-implantation, coinciding with a highly hypoxic NSC graft environment. Subsequently, strong spatiotemporal microglial and astroglial cell responses were initiated, which stabilised by day 5 post-implantation and remained present during the whole observation period. Moreover, the increase in astrocyte density was associated with a high degree of astroglial scarring within and surrounding the graft site. During the two-week follow up in this study, the NSC graft site underwent extensive remodelling with NSC graft survival further declining to around 1% of the initial number of grafted cells. CONCLUSIONS: The present study quantitatively describes the early post-transplantation events following NSC grafting in the adult mouse brain and warrants that such intervention is directly associated with a high degree of cell loss, subsequently followed by strong glial cell responses.
Assuntos
Células-Tronco Neurais/citologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Sobrevivência Celular , Células Cultivadas , Sobrevivência de Enxerto , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imunidade Inata , Camundongos , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/transplante , Análise Espaço-TemporalRESUMO
During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s). Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting. BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin). MRI on the other hand is a non-invasive technique which is clinically applicable and can be used to precisely locate cellular grafts with very high resolution, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies. In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).
Assuntos
Sistema Nervoso Central/citologia , Sistema Nervoso Central/cirurgia , Medições Luminescentes/métodos , Imageamento por Ressonância Magnética/métodos , Transplante de Células-Tronco , Animais , Compostos Férricos/análise , Sobrevivência de Enxerto , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Luciferases de Vaga-Lume/análise , Luciferases de Vaga-Lume/biossíntese , Luciferases de Vaga-Lume/genética , CamundongosRESUMO
Cell transplantation has been suggested to display several neuroprotective and/or neuroregenerative effects in animal models of central nervous system (CNS) trauma. However, while most studies report on clinical observations, currently little is known regarding the actual fate of the cell populations grafted and whether or how the brain's innate immune system, mainly directed by activated microglia and astrocytes, interacts with autologous cellular implants. In this study, we grafted well-characterized neural stem cell, mouse embryonic fibroblast, dendritic cell, bone marrow mononuclear cell, and splenocyte populations, all isolated or cultured from C57BL/6-eGFP transgenic mice, below the capsula externa (CE) of healthy C57BL/6 mice and below the inflamed/demyelinated CE of cuprizone-treated C57BL/6 mice. Two weeks postgrafting, an extensive quantitative multicolor histological analysis was performed in order (i) to quantify cell graft localization, migration, survival, and toxicity and (ii) to characterize endogenous CNS immune responses against the different cell grafts. Obtained results indicate dependence on the cell type grafted: (i) a different degree of cell graft migration, survival, and toxicity and (ii) a different organization of the endogenous immune response. Based on these observations, we warrant that further research should be undertaken to understand-and eventually control-cell graft-induced tissue damage and activation of the brain's innate immune system. The latter will be inevitable before cell grafting in the CNS can be performed safely and successfully in clinical settings.
Assuntos
Movimento Celular/fisiologia , Transplante de Células-Tronco , Traumatismos do Sistema Nervoso/patologia , Traumatismos do Sistema Nervoso/cirurgia , Animais , Células Cultivadas , Sistema Nervoso Central/citologia , Sistema Nervoso Central/cirurgia , Feminino , Sobrevivência de Enxerto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise de SobrevidaRESUMO
PURPOSE: Development of multimodal imaging strategies is currently of utmost importance for the validation of preclinical stem cell therapy studies. PROCEDURES: We performed a combined labeling strategy for bone marrow-derived stromal cells (BMSC) based on genetic modification with the reporter genes Luciferase and eGFP (BMSC-Luc/eGFP) and physical labeling with blue fluorescent micron-sized iron oxide particles (MPIO) in order to unambiguously identify BMSC localization, survival, and differentiation following engraftment in the central nervous system of mice by in vivo bioluminescence (BLI) and magnetic resonance imaging and postmortem histological analysis. RESULTS: Using this combination, a significant increase of in vivo BLI signal was observed for MPIO-labeled BMSC-Luc/eGFP. Moreover, MPIO labeling of BMSC-Luc/eGFP allows for the improved identification of implanted cells within host tissue during histological observation. CONCLUSIONS: This study describes an optimized labeling strategy for multimodal stem cell imaging resulting in improved quantitative and qualitative detection of cellular grafts.
Assuntos
Células da Medula Óssea/citologia , Compostos Férricos/química , Proteínas de Fluorescência Verde/metabolismo , Imageamento Tridimensional/métodos , Luciferases/metabolismo , Tamanho da Partícula , Coloração e Rotulagem , Animais , Fluorescência , Imageamento por Ressonância Magnética , Masculino , Camundongos , Células Estromais/citologia , Células Estromais/transplanteRESUMO
While neural stem cells (NSCs) are widely expected to become a therapeutic agent for treatment of severe injuries to the central nervous system (CNS), currently there are only few detailed preclinical studies linking cell fate with experimental outcome. In this study, we aimed to validate whether IV administration of allogeneic NSC can improve experimental autoimmune encephalomyelitis (EAE), a well-established animal model for human multiple sclerosis (MS). For this, we cultured adherently growing luciferase-expressing NSCs (NSC-Luc), which displayed a uniform morphology and expression profile of membrane and intracellular markers, and which displayed an in vitro differentiation potential into neurons and astrocytes. Following labeling with green fluorescent micron-sized iron oxide particles (f-MPIO-labeled NSC-Luc) or lentiviral transduction with the enhanced green fluorescent protein (eGFP) reporter gene (NSC-Luc/eGFP), cell implantation experiments demonstrated the intrinsic survival capacity of adherently cultured NSC in the CNS of syngeneic mice, as analyzed by real-time bioluminescence imaging (BLI), magnetic resonance imaging (MRI), and histological analysis. Next, EAE was induced in C57BL/6 mice followed by IV administration of NSC-Luc/eGFP at day 7 postinduction with or without daily immunosuppressive therapy (cyclosporine A, CsA). During a follow-up period of 20 days, the observed clinical benefit could be attributed solely to CsA treatment. In addition, histological analysis demonstrated the absence of NSC-Luc/eGFP at sites of neuroinflammation. In order to investigate the absence of therapeutic potential, BLI biodistribution analysis of IV-administered NSC-Luc/eGFP revealed cell retention in lung capillaries as soon as 1-min postinjection, resulting in massive inflammation and apoptosis in lung tissue. In summary, we conclude that IV administration of NSCs currently has limited or no therapeutic potential for neuroinflammatory disease in mice, and presumably also for human MS. However, given the fact that grafted NSCs have an intrinsic survival capacity in the CNS, their therapeutic exploitation should be further investigated, and-in contrast to several other reports-will most likely be highly complex.
Assuntos
Encefalomielite Autoimune Experimental/terapia , Células-Tronco Neurais/transplante , Animais , Células Cultivadas , Sistema Nervoso Central/patologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/patologia , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imunossupressores/farmacologia , Imunossupressores/uso terapêutico , Injeções Intravenosas , Luciferases/genética , Luciferases/metabolismo , Medições Luminescentes , Camundongos , Camundongos Transgênicos , Células-Tronco Neurais/efeitos dos fármacos , Transplante HomólogoRESUMO
Although adult and embryonic stem cell-based therapy for central nervous system (CNS) injury is being developed worldwide, less attention is given to the immunological aspects of allogeneic cell implantation in the CNS. The latter is of major importance because, from a practical point of view, future stem cell-based therapy for CNS injury will likely be performed using well-characterised allogeneic stem cell populations. In this study, we aimed to further describe the immunological mechanism leading to rejection of allogeneic bone marrow-derived stromal cells (BM-SC) after implantation in murine CNS. For this, we first investigated the impact of autologous and allogeneic BM-SC on microglia activation in vitro. Although the results indicate that both autologous and allogeneic BM-SC do not activate microglia themselves in vitro, they also do not inhibit activation of microglia after exogenous stimuli in vitro. Next, we investigated the impact of allogeneic BM-SC on microglia activation in vivo. In contrast to the in vitro observations, microglia become highly activated in vivo after implantation of allogeneic BM-SC in the CNS of immune-competent mice. Moreover, our results suggest that microglia, rather than T-cells, are the major contributors to allograft rejection in the CNS.