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Title: CONTINUOUS CULTURES OF MACROPHAGES DERIVED FROM THE 8-DAY EPIBLAST OF THE PIG.

Author
item Talbot, Neil
item WORKU, MULUMEBET - FDA
item Paape, Max
item Grier, Patricia
item Rexroad Jr, Caird
item Pursel, Vernon

Submitted to: In Vitro Cellular And Developmental Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/14/1995
Publication Date: N/A
Citation: N/A

Interpretive Summary: Eight-days after fertilization embryos of the pig were were collected before they implanted into the uterus. The epiblast tissue which gives rise to all the tissues of the body during embryonic development was isolated from the eight-day post-fertilization pig embryos and grown in vitro. Among the various cells that differentiated from the growing pig epiblast tissue were round, ameboid cells that resembled macrophages, the white blood cells in the body tissues which fight bacteria. Several tests and assays were conducted to prove that the pig epiblast derived macrophage-like (EDM) cells were indeed macrophages. The EDM cells were positive for reaction with several macrophage specific antibodies. EDM cells ingested bacteria with high efficiency like macrophages would. Electron microscopic analysis showed the EDM cells to have ultrastructural features typical of macrophages. Also typical of macrophages, the EDM cells bound acetylated-LDL, and produced tumor necrosis factor-like activity. This study proves that pig epiblast tissue is pluripotent in vitro. Also, because macrophages from the body are generally believed to be impossible to grow in vitro, the epiblast-derived macrophages are a unique reagent that may be useful in research and clinical applications.

Technical Abstract: Secondary macrophage cell cultures were generated from the primary culture ofe piblasts of 8-day old pig blastocysts. The epiblast-derived macrophage like (EDM) cells have a morphology and ameboid behavior that is typical of tissue histocytes. The cells reacted positively with monoclonal antibodies specific for pig granulocyte-macrophage lineage cells, & were not reactive with monoclonal antibodies specific for pig B and T lymphocytes. Marked phagocytic behavior and the formation of phagosomes were demonstrated following incubation with FITC-labeled bacteria. The EDM cells stained positively for non-specific acid esterase that was not inhibited by sodium fluoride. DiI-acetylated-LDL was rapidly taken up by the cells. Trans- mission electron microscopy of the EDM cells showed phagolysosomes, numerous cytoplasmic vacuoles, large, lobed nuclei, & numerous pseudopods or filopodia at the cell surface. Strong reactivity of the cells with anti-CD-14 monoclonal antibody was observed. Further, cytotoxic activity was produced from the EDM cells after exposure to lipopolysaccharide in a concentration and time dependent manner. The cultures could be maintained and expanded for several months on STO co-culture. Their derivation from the epiblast of the pig demonstrates the possibility of obtaining hemopoietic cell cultures from the preimplantation blastocysts of all mammals.