Microglial precursors derived from mouse embryonic stem cells

Abstract

Microglia are resident immune cells of the central nervous system. They can be directly isolated from the brain or from mixed postnatal glial cultures. Isolation of primary microglia is inefficient due to low yield. The cell line BV2 was used as a substitute for primary microglia, but BV2 are oncogenically transformed cells. Here, we established a protocol to generate microglial precursor lines from mouse embryonic stem (ES) cells. Microglial precursor cells were obtained from murine ES cells by differentiation of embryoid bodies to microglia within a mixed brain culture. Several independent ES cell-derived microglial precursor (ESdM) lines were generated and characterized by flow cytometry, immunocytochemistry, and functional assays. All ESdM showed expression of IBa1, CD11b, CD45, F4/80, CD49d, and CD29, but were negative for cKit and CD34. Stimulation with interferon-gamma or lipopolysaccharide (LPS) demonstrated upregulation of proinflammatory cytokine gene transcription including nitric oxide synthase-2, interleukin-1beta, and tumor necrosis factor-alpha at levels comparable to primary microglia. The ESdM showed efficient and rapid phagocytosis of microsphere beads, which was increased after stimulation with LPS. ESdM expressed the chemokine receptor CX3CR1 and demonstrated directed migration toward the ligand CX3CL1. After in vivo transplantation into postnatal brain tissue, ESdM showed engraftment as cells with a microglial phenotype and morphology. Thus, ESdM are stable proliferating cells substantially having most characteristics of primary microglia and therefore being a suitable tool to study microglial function in vitro and in vivo.

Fractalkine Attenuates Excito-neurotoxicity via Microglial Clearance of Damaged Neurons and Antioxidant Enzyme Heme Oxygenase-1 Expression

“…We developed a novel microglial phagocytosis assay using primary mouse cortical neurons; microglial phagocytosis has been previously assessed by uptake of fluorescent beads (30,31). Mouse primary cortical neurons were labeled with fluorescent DiI, and treated with 10 M glutamate for 24 h to induce damage.…”

GFAP‐independent inflammatory competence and trophic functions of astrocytes generated from murine embryonic stem cells

“…Generation of microglial precursor cells from murine embryonic stem cells has recently been described under neural-inductive conditions, and via nestin-postive intermediate stages (Napoli et al, 2009). Therefore, we examined the potential presence of microglial cells.…”

Resident macrophages in the cochlear blood-labyrinth barrier and their renewal via migration of bone-marrow-derived cells

“…F4/80 is a 160-kDa cell-surface glycoprotein expressed on a wide range of mature tissue macrophages, including Kupffer and Langerhans cells, microglia, and macrophages (Peng and Murr 2007;Iijima et al 2007;Napoli et al 2009). CD68 is a 110-kDa transmembrane glycoprotein, which is highly expressed by human monocytes and tissue macrophages (Pilling et al 2009;Soulas et al 2009).…”

“…1). The cells were verified as being resident PVMs by positive immunolabeling for specific cell surface markers (Peng and Murr 2007;Iijima et al 2007;Napoli et al 2009;Pilling et al 2009;Soulas et al 2009), including F4/80, CD68, CD11b (Fig. 1a-c), and MOMA2 (data not shown).…”