ABSTRACT
We document the protocols and methods for the production of immortalized cell lines of human neural stem cells from the human fetal central nervous system (CNS) cells by using a retroviral vector encoding v-myc oncogene. One of the human neural stem cell lines (HB1.F3) was found to express nestin and other specific markers for human neural stem cells, giving rise to three fundamental cell types of the CNS: neurons, astrocytes, and oligodendrocytes. After transplantation into the brain of mouse model of stroke, implanted human neural stem cells were observed to migrate extensively from the site of implantation into other anatomical sites and to differentiate into neurons and glial cells.
Subject(s)
Cell Culture Techniques/methods , Cell Differentiation , Central Nervous System/cytology , Multipotent Stem Cells/cytology , Cell Line , Cell Line, Transformed , Cells, Cultured , Cytogenetic Analysis , Fetus/cytology , Gene Expression Regulation , Gene Transfer Techniques , Humans , Immunoblotting , Immunohistochemistry , Multipotent Stem Cells/metabolism , Organ Specificity , Retroviridae , Reverse Transcriptase Polymerase Chain Reaction , beta-Galactosidase/metabolismABSTRACT
The expression of cytokines and cytokine receptors was investigated in enriched populations of human fetal Schwann cells by reverse transcribed-PCR and enzyme-linked immunosorbent assay. Human fetal Schwann cells constitutively expressed mRNA of IL-1beta, IL-6, IL-8, IL-11, IL-12, IL-15 and TGF-beta, and also cytokine receptors for IL-1, IL-4, IL-6, IL-8, IL-13, tissue necrosis factor and gp130. The expression of IL-1beta, IL-6 and IL-15 was upregulated following treatment with IL-1beta or TGF-beta. The protein levels of IL-6 were increased with IL-1beta treatment, but were decreased with IFN-gamma treatment. Human Schwann cells may respond to cytokine signals in the nerve injury sites and modify the pathological conditions by secreting cytokines. The secreted cytokines may play a role in leukocyte recruitment and exacerbation of axonal injury process.
Subject(s)
Cytokines/metabolism , Receptors, Cytokine/metabolism , Schwann Cells/metabolism , Cells, Cultured , Cytokines/genetics , Cytokines/pharmacology , Enzyme-Linked Immunosorbent Assay/methods , Ganglia, Spinal/cytology , Humans , RNA, Messenger/metabolism , Receptors, Cytokine/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Up-Regulation/drug effects , Up-Regulation/physiologyABSTRACT
The present study examined the effects of ATP on the striatum of Sprague-Dawley rats. Intrastriatal administration of ATP produced dose-dependent striatal lesions as confirmed by cresyl violet staining. Additional immunostaining using neuronal nuclear protein (NeuN), OX-42 and GFAP antibodies revealed that ATP caused death of both neurons and glial cells. The nonmetabolizable ATP analogue ATPgammaS and P2X receptor agonist alpha,beta-methylene ATP (alpha,beta-MeATP) mimicked ATP effects, whereas either P2Y receptor agonist ADP or P1 receptor agonist adenosine did not. The P2 receptor antagonist reactive blue 2, but not pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) attenuated ATP-induced striatal injury. These results suggest that intrastriatal administration of ATP causes P2X receptor-mediated cell death in the striatum and support the hypothesis that extracellular ATP can be an important mediator of neuropathological events of brain injuries.
Subject(s)
Adenosine Triphosphate/metabolism , Antigens, CD , Antigens, Neoplasm , Antigens, Surface , Avian Proteins , Blood Proteins , Hypoxia-Ischemia, Brain/metabolism , Neostriatum/metabolism , Neuroglia/metabolism , Neurons/metabolism , Neurotoxins/metabolism , Receptors, Purinergic P2/metabolism , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/pharmacology , Animals , Astrocytes/drug effects , Astrocytes/metabolism , Astrocytes/pathology , Basigin , Cell Death/drug effects , Cell Death/physiology , Dose-Response Relationship, Drug , Female , Glial Fibrillary Acidic Protein/metabolism , Hypoxia-Ischemia, Brain/physiopathology , Membrane Glycoproteins/metabolism , Microglia/drug effects , Microglia/metabolism , Microglia/pathology , Neostriatum/drug effects , Neostriatum/physiopathology , Nerve Degeneration/chemically induced , Nerve Degeneration/metabolism , Nerve Degeneration/physiopathology , Neuroglia/drug effects , Neuroglia/pathology , Neurons/drug effects , Neurons/pathology , Neurotoxins/toxicity , Purinergic P1 Receptor Agonists , Purinergic P1 Receptor Antagonists , Purinergic P2 Receptor Agonists , Purinergic P2 Receptor Antagonists , Rats , Rats, Sprague-Dawley , Receptors, Purinergic P1/metabolism , Receptors, Purinergic P2XABSTRACT
Enriched populations of human microglial cells were isolated from mixed cell cultures prepared from embryonic human telencephalon tissues. Human microglial cells exhibited cell type-specific antigens for macrophage-microglia lineage cells including CD11b (Mac-1), CD68, B7-2 (CD86), HLA-ABC, HLA-DR and ricinus communis aggulutinin lectin-1 (RCA-1), and actively phagocytosed latex beads. Gene expression and protein production of cytokines, chemokines and cytokine/chemokine receptors were investigated in the purified populations of human microglia. Normal unstimulated human microglia expressed constitutively mRNA transcripts for interleukin- 1beta (IL-1beta) -6, -8, -10, -12, -15, tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and monocyte chemoattractant protein-1 (MCP-1), while treatment with lipopolysaccharide (LPS) or amyloid beta peptides (Abeta) led to increased expression of mRNA levels of IL-8, IL-10, IL-12, TNF-alpha, MIP-1alpha, MIP-1beta, and MCP-1. Human microglia, in addition, expressed mRNA transcripts for IL-1RI, IL-1RII, IL-5R, IL-6R, IL-8R, IL-9R, IL-10R, IL-12R, IL-13R, and IL-15R. Enzyme-linked immunosorbent assays (ELISA) showed increased protein levels in culture media of IL-1beta, IL-8, TNF-alpha, and MIP-1alpha in human microglia following treatment with LPS or Abeta. Increased TNF-alpha release from human microglia following LPS treatment was completely inhibited with IL-10 pretreatment, but not with IL-6, IL-9, IL-12, IL-13, or transforming growth factor-beta (TGF-beta). Present results should help in understanding the basic microglial biology, but also the pathophysiology of activated microglia in neurological diseases such as Alzheimer disease, Parkinson disease, Huntington disease, amyotrophic lateral sclerosis, stroke, and neurotrauma.
Subject(s)
Chemokines/analysis , Cytokines/analysis , Microglia/metabolism , Receptors, Cytokine/analysis , Brain/cytology , Brain/embryology , Cells, Cultured , Chemokines/biosynthesis , Chemokines/genetics , Cytokines/biosynthesis , Cytokines/genetics , Enzyme-Linked Immunosorbent Assay , Gene Expression Regulation, Developmental , Humans , Microglia/cytology , Receptors, Cytokine/biosynthesis , Receptors, Cytokine/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
We recently showed that trisialoganglioside (GT1b) induces cell death of dopaminergic neurons in rat mesencephalic cultures (Chung et al., Neuroreport 12:611-614, 2001). The present study examines the in vivo neurotoxic effects of GT1b on dopaminergic neurons in the substantia nigra (SN) of Sprague-Dawley rats. Seven days after GT1b injection into the SN, immunocytochemical staining of SN tissue revealed death of nigral neurons, including dopaminergic neurons. Additional immunostaining using OX-42 and OX-6 antibodies showed that GT1b-activated microglia were present in the SN where degeneration of nigral neurons was found. Western blot analysis and double-labeled immunohistochemistry showed that inducible nitric oxide synthase (iNOS) was expressed in the SN, where its levels were maximal at 8 h post-GT1b injection, and that iNOS was localized exclusively within microglia. GT1b-induced loss of dopaminergic neurons in the SN was partially inhibited by N(G)-nitro-L-arginine methyl ester hydrochloride, an NOS inhibitor. Our results indicate that in vivo neurotoxicity of GT1b against nigral dopaminergic neurons is at least in part mediated by nitric oxide released from activated microglia. Because GT1b exists abundantly in central nervous system neuronal membranes, our data support the hypothesis that immune-mediated events triggered by endogenous compounds such as GT1b could contribute to the initiation and/or the progression of dopaminergic neuronal cell death that occurs in Parkinson's disease.
