Single cell suspensions, prepared from brain stem, cerebellum, and forebrain parenchyma ofembryonic and adult mice, were plated on monolayers of an astroglial cell line derived from a spontaneously immortalized mouse cerebellar culture, the Di9 clone. A few of the brain cells adhering to the Di9 monolayers were immunoreactive to the Mac-i antibody, which labels all cells of the monocytic and granulocytic lineages. The Mac-l-positive cells proliferated vigorously and later most of them acquired the F4/80 epitope specific for macrophages and microglia cells. Studies in clonal conditions allowed development oflarge colonies ofabout 2 x 105 cells that expressed typical microglia markers. Bone marrow Mac-ipositive cells cocultured on Di9 monolayers were also induced to proliferate, whereas peritoneal macrophages were not. Di9 astrocytes express macrophage colony-stimulating factor (CSF-1) activity at a high level, and their conditioned media induced the proliferation of brain and bone marrow Mac-ipositive cells. A specific anti-CSF-i antiserum completely blocked bone marrow macrophage progenitor proliferation and significantly reduced the multiplication of microglial precursors induced by the Di9-conditioned medium. These data indicate that the embryonic and adult mouse brain parenchyma contains potential progenitors for microglial cells.Microglia, first described by del Rio Hortega (1), form a distinct population of glial cells in the central nervous system (CNS). Recent reports strongly suggest that microglia are specialized cells of the mononuclear phagocyte lineage. Indeed, it has been shown that cells morphologically similar to those originally described as microglial in the brain parenchyma ofdeveloping and adult mice are labeled by monocyteand macrophage-specific markers. Microglia express FcR, type 3 complement receptor, and a macrophage-specific membrane glycoprotein of unknown function, recognized by the 2.4G2, Mac-1, and F4/80 monoclonal antibodies (2), respectively. Thus, it appears that brain microglia, like the other resident macrophages, could be the progeny of hemopoietic bone marrow progenitors. However, it has not been established whether the progenitors are present in the CNS before the formation of the blood-brain and meningeal barriers or if they can penetrate the brain during the adult life of normal mice to replace the decaying microglial cells. It is also known that cells that multiply in vivo after a brain injury (3) or in vitro in cultures of cerebral hemispheres from perinatal rat (4) express microglial markers; however, these studies did not indicate the actual number of cell doublings, and they have not determined whether the multiplying cells are microglial cells or their progenitors.In this study, we have investigated the presence and the proliferative capacity of microglial progenitors in different regions ofthe developing and adult mouse brain parenchyma.To this end, we have used an experimental system whereby brain cells were cultured on monolayers of the D19 astroglial cell ...
