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Isolation of large numbers of enriched human megakaryocytes from liquid
cultures of normal peripheral blood progenitor cells
EM Mazur, D Basilico, JL Newton, JL Cohen, C Charland, PA Sohl and A Narendran
Department of Medicine, Miriam Hospital, Providence, RI 02906.
Investigations linking human megakaryocyte development and cell biology
have been hindered by an inability to obtain large, relatively pure
megakaryocyte cell preparations from in vitro stem cell cultures. We report
here that such preparations can be generated from liquid cultures of normal
human peripheral blood mononuclear cells stimulated by a serum source of
megakaryocyte colony stimulating activity (Meg- CSA, the 0% to 60% ammonium
sulfate protein fraction of aplastic canine serum). Adherent-depleted
peripheral blood mononuclear cells are suspended at 5 x 10(5) to 10(6)
cells/mL in supplemented liquid culture medium, platelet-poor human plasma
20% (vol/vol) and 1 to 2 mg/mL serum Meg-CSA protein. After 12 to 14 days
of incubation, megakaryocytes constitute 3.0 +/- 2.9% (mean +/- SD, n = 8)
of the unseparated cultured cell population. Megakaryocytes can be enriched
by counterflow centrifugal elutriation to a purity of 58 +/- 14% (+/- SD)
with a recovery of 13 +/- 7% and a viability of 67 +/- 19%. This algorithm
results in the average isolation of approximately 3 x 10(5) enriched
megakaryocytes from a 100-mL starting volume of peripheral blood. Cultured
megakaryocytes exhibit normal light and ultrastructural morphology by
Wright-Giemsa staining and electron microscopic analysis. After a 12-day
culture interval, enriched megakaryocyte preparations exhibit morphologic
stage distributions that are similar to normal human marrow. Stage
distributions move rightward with culture duration indicating partial
synchrony of megakaryocyte maturation. On cytospin preparations,
megakaryocyte diameter averages 30.2 +/- 1.5 microns and increases with
maturation stage. Flow cytometric analyses demonstrate the expression of
platelet glycoproteins (GP) Ib and IIb/IIIa by the cultured megakaryocytes.
The modal ploidy of the enriched cells at day 12 of culture is 16N and most
remaining megakaryocytes are 8N or 32N. Liquid culture of serum
Meg-CSA-stimulated human peripheral blood mononuclear cells represents a
valuable investigative tool that should permit studies of human
megakaryocyte biology that have not been possible in the past.
Volume 76,
Issue 9,
pp. 1771-1782,
11/01/1990
Copyright © 1990 by The American Society of Hematology
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