RESUMEN
Astrocytes are the most abundant cell type of the central nervous system and cover a broad range of functionalities. We report here the generation of a novel monoclonal antibody, anti-astrocyte cell surface antigen-2 (Anti-ACSA-2). Flow cytometry, immunohistochemistry and immunocytochemistry revealed that Anti-ACSA-2 reacted specifically with a not yet identified glycosylated surface molecule of murine astrocytes at all developmental stages. It did not show any labeling of non-astroglial cells such as neurons, oligodendrocytes, NG2+ cells, microglia, endothelial cells, leukocytes, or erythrocytes. Co-labeling studies of GLAST and ACSA-2 showed largely overlapping expression. However, there were also notable differences in protein expression levels and frequencies of single-positive subpopulations of cells in some regions of the CNS such as cerebellum, most prominently at early postnatal stages. In the neurogenic niches, the dentate gyrus of the hippocampus and the subventricular zone (SVZ), again a general overlap with slight differences in expression levels were observed. ACSA-2 was unlike GLAST not sensitive to papain-based tissue dissociation and allowed for a highly effective, acute, specific, and prospective purification of viable astrocytes based on a new rapid sorting procedure using Anti-ACSA-2 directly coupled to superparamagnetic MicroBeads. In conclusion, ACSA-2 appears to be a new surface marker for astrocytes, radial glia, neural stem cells and bipotent glial progenitor cells which opens up the possibility of further dissecting the characteristics of astroglial subpopulations and lineages.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos de Superficie/análisis , Antígenos de Superficie/inmunología , Astrocitos/citología , Astrocitos/inmunología , Separación Inmunomagnética/métodos , Animales , Animales Recién Nacidos , Especificidad de Anticuerpos , Antígenos de Superficie/metabolismo , Encéfalo/citología , Encéfalo/crecimiento & desarrollo , Células Cultivadas , Células Endoteliales/citología , Células Endoteliales/inmunología , Eritrocitos/citología , Eritrocitos/metabolismo , Transportador 1 de Aminoácidos Excitadores/análisis , Leucocitos/citología , Leucocitos/inmunología , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/citología , Microglía/inmunología , Células-Madre Neurales/inmunología , Neuronas/citología , Neuronas/metabolismo , Oligodendroglía/citología , Oligodendroglía/inmunología , Ratas WistarRESUMEN
The macrophage migration inhibitory factor (MIF) receptor CD74 is overexpressed in various neoplasms, mainly in hematologic tumors, and currently investigated in clinical studies. CD74 is quickly internalized and recycles after antibody binding, therefore it constitutes an attractive target for antibody-based treatment strategies. CD74 has been further described as one of the most up-regulated molecules in human glioblastomas. To assess the potential relevance for anti-CD74 treatment, we determined the cellular source and clinicopathologic relevance of CD74 expression in human gliomas by immunohistochemistry, immunofluorescence, immunoblotting, cell sorting analysis and quantitative polymerase chain reaction (qPCR). Furthermore, we fractionated glioblastoma cells and glioma-associated microglia/macrophages (GAMs) from primary tumors and compared CD74 expression in cellular fractions with whole tumor lysates. Our results show that CD74 is restricted to GAMsâ in vivo, while being absent in tumor cells, the latter strongly expressing its ligand MIF. Most interestingly, a higher amount of CD74-positive GAMs was associated with beneficial patient survival constituting an independent prognostic parameter and with an anti-tumoral M1 polarization. In summary, CD74 expression in human gliomas is restricted to GAMs and positively associated with patient survival. In conclusion, CD74 represents a positive prognostic marker most probably because of its association with an M1-polarized immune milieu in high-grade gliomas.
Asunto(s)
Neoplasias Encefálicas , Antígeno CD47/metabolismo , Glioma , Macrófagos/metabolismo , Microglía/metabolismo , Regulación hacia Arriba/fisiología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Antígeno CD47/genética , Proteínas de Unión al Calcio , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Femenino , Citometría de Flujo , Proteína Ácida Fibrilar de la Glía/metabolismo , Glioma/metabolismo , Glioma/mortalidad , Glioma/patología , Humanos , Estimación de Kaplan-Meier , Masculino , Análisis por Micromatrices , Proteínas de Microfilamentos , ARN Mensajero/metabolismoRESUMEN
Astrocytes show large morphological and functional heterogeneity and are involved in many aspects of neural function. Progress in defining astrocyte subpopulations has been hampered by the lack of a suitable antibody for their direct detection and isolation. Here, we describe a new monoclonal antibody, ACSA-1, which was generated by immunization of GLAST1 knockout mice. The antibody specifically detects an extracellular epitope of the astrocyte-specific L-glutamate/L-aspartate transporter GLAST (EAAT1, Slc1a3). As shown by immunohistochemistry, immunocytochemistry, and flow cytometry, ACSA-1 was cross-reactive for mouse, human, and rat. It labeled virtually all astrocytes positive for GFAP, GS, BLBP, RC2, and Nestin, including protoplastic, fibrous, and reactive astrocytes as well as Bergmann glia, Müller glia, and radial glia. Oligodendrocytes, microglia, neurons, and neuronal progenitors were negative for ACSA-1. Using an immunomagnetic approach, we established a method for the isolation of GLAST-positive cells with high purity. Binding of the antibody to GLAST and subsequent sorting of GLAST-positive cells neither interfered with cellular glutamate transport nor compromised astrocyte viability in vitro. The ACSA-1 antibody is not only a valuable tool to identify and track astrocytes by immunostaining, but also provides the possibility of separation and further analysis of pure astrocytes.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Astrocitos/metabolismo , Encéfalo/citología , Transportador 1 de Aminoácidos Excitadores/inmunología , Transportador 1 de Aminoácidos Excitadores/metabolismo , Animales , Animales Recién Nacidos , Ácido Ascórbico , Ácido Aspártico/metabolismo , Encéfalo/metabolismo , Antígeno CD11b/metabolismo , Células Cultivadas , Electroporación/métodos , Transportador 1 de Aminoácidos Excitadores/deficiencia , Transportador 1 de Aminoácidos Excitadores/farmacología , Femenino , Citometría de Flujo , Gangliósidos/metabolismo , Glutamato-Amoníaco Ligasa/metabolismo , Humanos , Magnesio , Ratones , Ratones Noqueados , Proteínas de la Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito , Proteínas del Tejido Nervioso/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Neuronas/metabolismo , Ratas , Ácidos Siálicos/metabolismo , Tritio/metabolismo , Vitamina B 6RESUMEN
Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads.
Asunto(s)
Encéfalo/citología , Técnicas Citológicas/métodos , Animales , Química Encefálica/fisiología , RatonesRESUMEN
Gamma-aminobutyric acid (GABA)ergic neurons are a diverse group of inhibitory neurons playing crucial roles in information processing. We analyzed the gene expression of regionally defined GABAergic neurons from the cortex, olfactory bulb, striatum, and cerebellum of glutamate decarboxylase 67-green fluorescence protein (GAD67-GFP) knock-in mice. We introduce a generally applicable method for singularization of brain cells, flow cytometric enrichment, and global mRNA amplification for sensitive gene expression profiling. Systematic quantification elicited a high dynamic range of GABAergic cell numbers in different brain regions. Clustering of our gene expression results revealed major differences between hind and forebrain GABAergic neurons indicating that the development of GABAergic neurons depends on their regional location. While GABAergic neurons of the forebrain are characterized by three main groups of transcription factors of the Distal-less-family, the POU-family, and ETS/FOX family, specific members of the ZIC- and LHX-family of transcription factors appear to define hindbrain inhibitory neurons.
Asunto(s)
Encéfalo/anatomía & histología , Neuronas/fisiología , Ácido gamma-Aminobutírico/metabolismo , Animales , Encéfalo/metabolismo , Análisis por Conglomerados , Perfilación de la Expresión Génica , Técnicas de Sustitución del Gen , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Neuronas/citología , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
The progeny of neural stem cells in the subventricular zone (SVZ) of the adult mammalian brain consists in polysialylated NCAM-expressing immature neurons (PSA(+) cells), which migrate to the olfactory bulb (OB) to differentiate into GABAergic interneurons. We purified murine PSA(+) cells directly from the adult brain by FACS and analyzed their gene expression profile by SAGE. Comparative analyses led to the identification of precursor-enriched genes, including Survivin, Sox-4, Meis2, Dishevelled-2, C3aR1 and Riken 3110003A17, and many so far uncharacterized transcripts. Cluster analysis showed that groups of genes involved in axon guidance and gene clusters implicated in chemotaxis are strongly upregulated, indicating a role of both cues in the control of cell migration in the adult brain. Furthermore, genes involved in apoptosis and cell proliferation are co-expressed, suggesting that the amount of precursors that is present in the adult brain is a result of an equilibrium of these processes.