Efficient technique for immortalization of murine microglial cells relevant for studies in murine models of multiple sclerosis (original) (raw)
Related papers
Immortalization and characterization of rat microglial cells
Neuropathology and Applied Neurobiology, 1995
Immortalization and characterization of rat microglial cells Microglial cell lines from rat brain were established by transfer of a temperature sensitive simian virus 40 large tumour antigen by means of a retrovirus. Four weeks after infection, colonies were generated in the presence of neomycin and granulocyte-macrophage colony stimulating factor (GM-CSF), and subsequently subcloned. Both bulk cell lines and clones proIiferate actively at 3 3°C. whereas the rate of division was significantly decreased at 39°C when the large T antigen is non-functional. At 39°C these cells take on the microglial phenotype as demonstrated by immunoreactivity to ED-1 (an intracellular antigen), OX-42 (complement type 3 receptor), W 3 / 2 5 (CD4 homologue), OX-6 (MHC class I1 antigen) Keywords: microglia, immortalization. SV-40 large T antigen, cell line, rat and OX-18 (MHC class I antigen). These cells are capable of active phagocytosis and retain these properties for 10-15 passages. Long-term culture of these lines and clones, greater than 15 passages, displayed a gradual down-regulation of all cell surface specific antigens that were not rescued by lipopolysaccharide (LPS), interferongamma (y-IFN), GM-CSF or colony-stimulating factor-1 (CSF-1). The expression of the SV-40 large T antigen was unaffected. These results demonstrate the feasibility of immortalizing short-term cell lines with the SV-40 large T antigen for their use in the characterization of microglial properties.
Neuroscience Letters, 1995
Four continuous cell lines of human microglial cells were obtained by transfection of enriched cultures of human embryonic brainderived macrophages with a plasmid encoding for the large T antigen of SV40. The transformed ceils had the macrophagic characteristics of adherence and intra-.cytoplasmic non-specific esterase activity. They could phagocytize zymosan particles but the phagocytic activity remained low. They expressed several macrophagic antigens but not the monocytic markers CD14, CD4, CD68/Ki-M6 and CD1 lc. The cells could be activated to express class II major histocompatibility complex antigens after interferon-y activation. Finally, interleukin-6 was produced spontaneously by the cells and this production was further increased after interleukin-la stimulation.
An immortalized cell line expresses properties of activated microglial cells
Journal of Neuroscience Research, 1992
Murine cultured microglial cells were immortalized after infection with a v-raf/v-myc recombinant retro-virus. This immortalized cell line (BV-2) shares properties with body macrophages with respect to the antigen profile, their phagocytic capacity and antimicrobial activity. BV-2 cells are not constitu-tively able to kill tumor cells in vitro, but acquire antitumor activity following an increase in [Ca++]i. BV-2 cells, like microglial cells, are however, distinct from peripheral macrophages by their expression of inwardly rectifying K+ cannels in concert with a Jack in outwardly rectifying K+ channels and the formation of spineous processes. The BV-2 cell line thus represents a suitable model for in vitro studies of activated microglial cells.
Generation and characterization of mouse microglial cell lines
Journal of Neuroimmunology, 1994
A murine cell line (MMGT1) has been established after transfection of primary microglial cell cultures with a v-myc-containing plasmid. This cell line was comparable with primary microglial cells with respect to morphology, presence of acetylated low density lipoprotein receptor, non-specific esterase, CD63, major histocompatibility complex antigens and CDll, and binding for Ricinus communis agglutinin. Primary microglia as well as MMGT1 cells were negative for glial fibrillary acidic protein. Different MMGT1 strains were obtained after subcloning, two of which resembled histiocytes (F4/80 and BM-8). These cell strains, MMGT12 and 16, were able to opsonize latex beads, and could be induced by endotoxins (LPS) to secrete TNF-a, IL-1, IL-6, TGF-/3, and EGF. The other subclones had intermediate (MCA519, ER-MP20) or mixed macrophage characteristics and did not react to endotoxin by an increase in TNF-a, IL-1, and TGF-/3. Our newly established murine microglia lines may prove to be useful models to study inflammation and repair in the brain.
Glia, 2014
Microglia are resident antigen-presenting cells in the central nervous system (CNS) that either suppress or promote disease depending on their activation phenotype and the microenvironment. Multiple sclerosis (MS) is a chronic inflammatory disease causing demyelination and nerve loss in the CNS, and experimental autoimmune encephalomyelitis (EAE) is an animal model of MS that is widely used to investigate pathogenic mechanisms and therapeutic effects. We isolated and cultured microglia from adult mouse brains and exposed them to specific combinations of stimulatory molecules and cytokines, the combination of IL-4, IL-10, and TGF-b yielding the optimal regime for induction of an immunosuppressive phenotype (M2). M2 microglia were characterized by decreased expression or production of CD86, PD-L1, nitric oxide, and IL-6, increased expression of PD-L2, and having a potent capacity to retain their phenotype on secondary proinflammatory stimulation. M2 microglia induced regulatory T cells, suppressed T-cell proliferation, and downmodulated M1-associated receptor expression in M1 macrophages. Myelin oligodendrocyte glycoprotein (MOG)-induced EAE was induced in DBA/1 mice and at different time points (0, 5, 12, or 15 days postimmunization) 3 3 10 5 M2 microglia were transferred intranasally. A single transfer of M2 microglia attenuated the severity of established EAE, which was particularly obvious when the cells were injected at 15 days postimmunization. M2 microglia-treated mice had reduced inflammatory responses and less demyelination in the CNS. Our findings demonstrate that adult M2 microglia therapy represents a novel intervention that alleviated established EAE and that this therapeutic principle may have relevance for treatment of MS patients.
An Overview of in vitro Methods to Study Microglia
Frontiers in Cellular Neuroscience
Neuroinflammation is a common feature in neurodegenerative diseases and strategies to modulate neuroinflammatory processes are increasingly considered as therapeutic options. In such strategies, glia cells rather than neurons represent the cellular targets. Microglia, the resident macrophages of the central nervous system, are principal players in neuroinflammation and detailed cellular biological knowledge of this particular cell type is therefore of pivotal importance. The last decade has shed new light on the origin, characteristics and functions of microglia, underlining the need for specific in vitro methodology to study these cells in detail. In this review we provide a comprehensive overview of existing methodology such as cell lines, stem cell-derived microglia and primary dissociated cell cultures, as well as discuss recent developments. As there is no in vitro method available yet that recapitulates all hallmarks of adult homeostatic microglia, we also discuss the advantages and limitations of existing models across different species.
Journal of Neuroimmunology, 1995
We developed a panel of non-virus transformed cell lines derived from individual microglial precursors residing in the brains of normal mice. These colony stimulating factor-l-dependent cell lines are B7-l + (CDSO), Mac-l +, Mac-2'. Mac-3+, CD45+, MHC class I +, colony stimulating factor-1 receptor +, and they ingest antibody-coated particles. However, the cell lines differ in their expression of B7-2 (CD86), F4/80, Ly-6C and MHC class II molecules. They also differ in their ability to constitutively process and present antigens to naive CD4+ and CD8+ T cells, memory CD4+ and CD8+, and in the manner by which interferon y modulates their antigen-presenting activities. These cell lines should be valuable as models for studies on the immunobiology of the microglia.
Culturing Microglia from the Neonatal and Adult Central Nervous System
Journal of Visualized Experiments, 2013
Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.
Development of a Chimeric Model to Study and Manipulate Human Microglia In Vivo
Neuron
iPSC-derived microglia offer a powerful tool to study microglial homeostasis and diseaseassociated inflammatory responses. Yet, microglia are highly sensitive to their environment, exhibiting transcriptomic deficiencies when kept in isolation from the brain. Furthermore, species-specific genetic variations demonstrate that rodent microglia fail to fully recapitulate the human condition. To address this, we developed an approach to study human microglia within a surrogate brain environment. Transplantation of iPSC-derived hematopoieticprogenitors into the postnatal brain of humanized, immune-deficient mice results in contextdependent differentiation into microglia and other CNS macrophages, acquisition of an ex vivo human microglial gene signature, and responsiveness to both acute and chronic insults. Most notably, transplanted microglia exhibit robust transcriptional responses to A-plaques that only partially overlap with that of murine microglia, revealing new, human-specific Aresponsive genes. We therefore propose that this chimeric model can provide a powerful new system to examine the in vivo function of patient-derived and genetically-modified microglia.