Microglial cells can be derived directly from the dissociated brain tissue by sorting procedures, from postnatal glial cultures by mechanic isolation or from pluripotent stem cells by differentiation. The detailed molecular phenotype of microglia from different sources is still unclear. Here, we performed a whole transcriptome analysis of flow cytometry-sorted microglia, primary postnatal cultured microglia, embryonic stem cell derived microglia (ESdM), and other cell types. Microglia and ESdM, both cultured in serum-free medium, were closely related to sorted microglia and showed a unique transcriptome profile, clearly distinct to other myeloid cell types, T cells, astrocytes, and neurons. ESdM and primary cultured microglia showed strong overlap in their transcriptome. Only 143 genes were differentially expressed between both cell types, mainly derived from immune-related genes with a higher activation status of proinflammatory and immune defense genes in primary microglia compared to ESdM. Flow cytometry analysis of cell surface markers CD54, CD74, and CD274 selected from the microarray confirmed the close phenotypic relation between ESdM and primary cultured microglia. Thus, assessment of genome-wide transcriptional regulation demonstrates that microglial cells are unique and clearly distinct from other macrophage cell types.

Microglia, the resident immune cells of the brain, are difficult to obtain in high numbers and purity using currently available methods; to date, microglia for experimental research are mainly isolated from the brain or from mixed glial cultures. In this paper, we describe a basic protocol for the in vitro differentiation of mouse embryonic stem (ES) cells into microglial precursor cells. Microglia are obtained by a protocol consisting of five stages: (i) cultivation of ES cells, (ii) formation and differentiation of embryoid bodies, (iii) differentiation into neuroectodermal lineage and isolation of myeloid precursor cells, (iv) differentiation into microglial precursor cells and (v) cultivation of ES cell-derived microglial precursors (ESdMs). The protocol can be completed in 60 d and results in stably proliferating ESdM lines, which show inducible transcription of inflammatory genes and cell marker expression comparable with primary microglia. Furthermore, ESdMs are capable of chemokine-directed migration and phagocytosis, which are major functional features of microglia.

A broad spectrum of diseases is characterized by myelin abnormalities and/or oligodendrocyte pathology. In most, if not all, of these diseases, early activation of microglia occurs. Our knowledge regarding the factors triggering early microglia activation is, however, incomplete. In this study, we used the cuprizone model to investigate the temporal and causal relationship of oligodendrocyte apoptosis and early microglia activation. Genome-wide gene expression studies revealed the induction of distinct chemokines, among them Cxcl10, Ccl2, and Ccl3 in cuprizone-mediated oligodendrocyte apoptosis. Early microglia activation was unchanged in CCL2- and CCL3-deficient knockouts, but was significantly reduced in CXCL10-deficient mice, resulting in an amelioration of cuprizone toxicity at later time points. Subsequent in vitro experiments revealed that recombinant CXCL10 induced migration and a proinflammatory phenotype in cultured microglia, without affecting their phagocytic activity or proliferation. In situ hybridization analyses suggest that Cxcl10 mRNA is mainly expressed by astrocytes, but also oligodendrocytes, in short-term cuprizone-exposed mice. Our results show that CXCL10 actively participates in the initiation of microglial activation. These findings have implications for the role of CXCL10 as an important mediator during the initiation of neuroinflammatory processes associated with oligodendrocyte pathology.

Sialic-acid-binding immunoglobulin-like lectin-h (Siglec-h) is a recently identified mouse-specific CD33-related Siglec that signals via DAP12/TYROBP. Expression of Siglec-h has been observed on plasmacytoid dendritic cells and microglia, but the ligand and the function of Siglec-h remained elusive. Here, we demonstrate gene transcription and protein expression of Siglec-h by mouse microglia after interferon-γ treatment or polarization into a M1-subtype. Microglial Siglec-h acted as phagocytosis receptor since targeting of microsphere beads to Siglec-h triggered their uptake into the microglia. The extracellular domain of Siglec-h protein bound to mouse glioma lines, but not to astrocytes or other normal mouse cells. Microglial cells stimulated to express Siglec-h engulfed intact glioma cells without prior induction of apoptosis and slightly reduced glioma cell number in culture. Phagocytosis of glioma cells by activated microglia was dependent on Siglec-h and its adapter molecule DAP12. Thus, data show that M1-polarized microglial cells can engulf glioma cells via a DAP12-mediated Siglec-h dependent mechanism.

A comprehensive proteomic approach was applied to investigate molecular events occurring upon inoculation of Medicago truncatula cell-suspension cultures with the oomycete root pathogen Aphanomyces euteiches. Establishment of an inoculation assay in the cell cultures allowed a direct comparison between proteins induced by elicitation with a crude culture extract of the oomycete and by inoculation with A. euteiches zoospores representing the natural infection carrier. Oxidative burst assays revealed responsiveness of the cell cultures for perception of elicitation and inoculation signals. The plant "elicitation proteome" resembles the "inoculation proteome" in early incubation stages and includes proteins induced following initial oxidative burst and defense reactions, but also proteins involved in the antioxidative system. However, approximately 2 days after incubation, the inoculation proteome differs drastically from the proteome of elicited cultures, where a cessation of responses assignable to A. euteiches elicitation occurred. The specific protein induction patterns of zoospore-inoculated cells appeared consistent with the protein induction identified in recent studies for an A. euteiches infection in planta and consist of three functional groups: i) pathogenesis-related proteins, ii) proteins associated with secondary phenylpropanoid or phytoalexin metabolism, and, particularly, iii) proteins assigned to carbohydrate metabolism and energy-related cellular processes. Phosphoproteomic analyses revealed consistent and specific activation of these defense-related pathways already at very early timepoints of inoculation, providing evidence that the identified protein profiles are representative for an established A. euteiches infection of M. truncatula.