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Maturation Rate of Mouse Neutrophilic Granulocytes: Acceleration by Retardation of Proliferation, but No Detectable Influence from G-CSF or Stromal Cells

^a Department of Physiology, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway;
^b Nycomed Drug Research, Oslo, Norway

Key Words. Hematopoietic cells • Bone marrow stromal cells • Proliferation • Maturation • G-CSF

Dr. Haakon B. Benestad, Department of Physiology, IMBA, UiO, P.O. Box 1103 Blindern, N-0317 Oslo, Norway.

Our purpose was to examine the possible influence of stromal and humoral mediators on granulocytic maturation rates. Sorted immature murine progenitor (Lin^–Sca-1⁺) cells were cultured in peritoneal diffusion chambers (DCs) with or without a confluent layer of irradiated bone marrow stromal cells on one of the micropore membrane walls. In other experiments, 10 µg/kg/d recombinant G-CSF (rhG-CSF) was administered continuously into DC host mice through s.c. implanted osmotic minipumps. Operationally, maturation rate was assessed as the ratio between the number of polymorphonuclear cells (PMN) and proliferative granulocytes (PG) in short-term cultures, based on the differential cell counts, and supported by flow cytometric measurement of a granulocytic differentiation marker; and by the emergence time of PMN in the DCs, obtained by extrapolation. Also, increased maturation is associated with increased cell density, as reflected by the positioning of the granulocytes during centrifugation in a discontinuous Percoll gradient. This method, as well as the conversion rate of ³H-thymidine labeled PG into the heavier non-PG maturational stages, were also used as indicators of maturation rate. After five, six, and seven days of culture in the peritoneal cavity, DC cells were harvested. Their proliferative status, based on measurement of incorporated bromodeoxyuridine, was determined, and their maturation rates were evaluated. Proliferation of immature granulocytic progenitor cells was apparently inhibited by direct contact with bone marrow stromal cells, and stimulated by G-CSF during the early stage of culturing. However, the subsequent maturation rate, which could be accelerated by increasing culture cellularity, thus decreasing PG proliferation rate, was not detectably influenced by either stromal cells or G-CSF.

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