| Co-Investigator(Kenkyū-buntansha) |
YAMAMOTO Haruyasu EHIME UNIVERSITY, SCHOOL OF MEDICINE, PROFESSOR, 医学部, 教授 (10092446)
IMAI Yoshinori EHIME UNIVERSITY, SCHOOL OF MEDICINE, ASSOCIATE PROFESSOR, 医学部, 助教授 (20270689)
OGATA Tadanori EHIME UNIVERSITY, SCHOOL OF MEDICINE, LECTURER, 医学部, 講師 (30291503)
SUZUKI Shunji UNIVERSITY OF YAMANASHI, INTERDISCIPLINARY GRADUATE SCHOOL OF MEDICINE AND ENGINEERING, ASSOCIATE PROFESSOR, 医学工学総合研究部, 助教授 (60372728)
高橋 寿明 愛媛大学, 医学部, 助手 (20363228)
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| Budget Amount *help |
¥13,800,000 (Direct Cost: ¥13,800,000)
Fiscal Year 2005: ¥4,500,000 (Direct Cost: ¥4,500,000)
Fiscal Year 2004: ¥9,300,000 (Direct Cost: ¥9,300,000)
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| Research Abstract |
Microglia are considered the only cell population of mesodermal origin in the brain, although their role is not fully understood. The present study demonstrated that rat primary microglial cells expressed nestin, A2B5, and O4 antigens, which are markers for oligodendrocyte precursor cells. Based on these findings, we investigated whether microglial cells generated neurons or macroglial cells. Purified microglial cells were cultured in the presence of 10% fetal bovine serum for 3 d, followed by culture in the presence of 70% serum for 2 d. During the two-step culture, microglial cells became highly proliferative and strongly expressed inhibitor of DNA binding (Id) genes, indicative of dedifferentiation of the cells. The dedifferentiated cells also expressed transcription factors that promote differentiation into neurons or macroglial cells. When the dedifferentiated cells were transferred into serum-free medium on poly-L-lysine-coated substrate, a substantial number of the cells rapidly
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turned into long process-bearing cells, which expressed microtubule-associated protein 2, synapsin I, neurofilament proteins, glial fibrillary acidic protein, or galactocerebroside. When microglial cells were fluorescently labelled through acetylated low-density lipoprotein (LDL) receptors or by a phagocytosis-dependent mechanism, fluorescence-bearing neurons, astrocytes, or oligodendrocytes were observed. Neurospheres, aggregates of neural stem cells, expressed Musashi 1 and epidermal growth factor receptor, but the microglia-derived cells did not. These results suggest a novel role of microglia as multipotential stem cells to give rise to neurons, astrocytes, or oligodendrocytes.
Such multipotent MG called promicroglioblasts (ProMGBs) formed cell aggregates, which generated cells with neuroectodermal phenotypes shortly after their transfer into serum-free medium. As revealed by immunohistochemistry, there were a few MG expressing NG2 chondroitin sulfate proteoglycan (NG2) in the neonatal rat brain. Primary culture from the neonatal brain contained NG2^+ MG, which appeared to be the source of NG2^+ ProMGB aggregates. The aggregates were MG marker^+/NG2^+/GFAP^+/NCAM^+/S^-100b^- and had alkaline phosphatase activity. The marked accumulation of NG2^+ MG was observed close to stab wounds made in the mature rat brain. The accumulated NG2^+ MG in the wound gradually decreased in number, but the cells persisted up to 150 days postlesioning. In addition, GFAP immunoreactivity increased markedly around the wound. The NG2^+ MG in the wounds separated with trypsin-EDTA formed NG2^+ aggregates in 70% serum-supplemented medium and then transformed into cells with neuroectodermal phenotypes in serum-free medium. Although it is difficult to separate viable neurons from mature brains, cells from stab wounds generated process-bearing b-tubulin III^+ cells in vitro easily. These data suggest that NG2^+ MG in normal developing or pathologic brains are involved in the genesis or regeneration of the brain. Less
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