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In vitro effects of hematopoietic growth factors on the proliferation,
endoreplication, and maturation of human megakaryocytes
N Debili, E Hegyi, S Navarro, A Katz, MA Mouthon, J Breton-Gorius and W Vainchenker
INSERM U.91 Hopital Henri Mondor, Creteil, France.
A liquid culture technique was used to study regulation of human
megakaryocytopoiesis in vitro. Low-density cells from adult bone marrow
were cultured in the presence of normal plasma, plasma from patients with
aplastic marrows (AP), recombinant human granulocyte-macrophage
colony-stimulating factor (rhGM-CSF) and interleukin-3 (IL-3).
Megakaryocytes (MK) were studied at day 10 of culture by a two-color
staining technique using a pool of monoclonal antibodies for their
identification and propidium iodide to label DNA. Their ploidy distribution
was analyzed by flow cytometry. In some experiments cytoplasmic maturation
was also studied by ultrastructural techniques. Normal plasma provides a
low number of MK with a ploidy distribution including 8 N and 16 N MK. AP
promoted in a dose-dependent manner proliferation of MK and some batches
favored endoreplication. This effect was clearly demonstrated when ploidy
distribution was compared between normal plasma and AP on parallel marrow
cultures. However, ploidy distribution was shifted toward low values
compared with uncultured MK. rhGM-CSF had no significant effect on these
two parameters. In contrast, rhIL-3 from 0.1 U/mL to 100 U/mL had a
proliferative effect but was unable to induce endoreplication. Furthermore,
when associated with AP it totally abrogated the effect of AP on
endoreplication because in most experiments more than 90% of MK were 2 N
and 4 N. This effect was also observed when rhIL-3 was added after 7 days
of culture (when it has little proliferative effects). Studies of the
maturation of MK grown with rhIL-3 indicate that the majority were small
mature cells synthesizing alpha-granules and demarcation membranes. The
effect of AP on MK proliferation and endoreplication was not related to
IL-6 because its IL-6 content was identical to that of normal plasma and
its neutralization did not modify these parameters. In conclusion, this
study indicates that liquid culture technique in association with flow
cytometry could be a powerful tool in identifying the humoral regulators of
human megakaryocytopoiesis.
Volume 77,
Issue 11,
pp. 2326-2338,
06/01/1991
Copyright © 1991 by The American Society of Hematology
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