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The development of human megakaryocytes: III. Development of mature
megakaryocytes from highly purified committed progenitors in synthetic
culture media and inhibition of thrombopoietin-induced polyploidization by
interleukin-3
A Dolzhanskiy, RS Basch and S Karpatkin
Department of Pathology, New York University Medical Center, NY 10016, USA.
Megakaryocyte (MK) progenitors, CD34+CD41+ cells, were isolated from human
bone marrow with a purity greater than 98% and a viability of 95%, using
affinity techniques with magnetic beads followed by fluorescence-activated
cell sorting. These cells were incubated in synthetic media containing the
cytokines thrombopoietin (TPO), interleukin-3 (IL-3), stem cell factor
(SCF), and IL-6, obviating the confounding effects of serum growth factors
or cytokine secretions of non-MK cells on MK maturation. MK number, MK
colony-forming units (CFU- MK), and MK ploidy and phenotype were examined
during 7 days in culture. TPO in serum-free cultures without any other
exogenously added cytokine supported MK growth and maturation. SCF
synergized with TPO to augment MK production and maturation and could
partially replace it under some conditions. Both TPO and IL-3 alone
increased MK number (12- and 5-fold, respectively) and CFU-MK
(approximately 15-fold each). SCF alone had no effect on MK proliferation
in the absence of TPO, but increased both MK number and CFU-MK by 1.5- to
2.0-fold in the presence of TPO. When combined with IL-3, SCF increased
both MK number and CFU- MK by 15- to 20-fold in the absence of TPO. In the
presence of TPO, the combination of IL-3 and SCF produced only modest
increases (1.5- to 2.0- fold) in both MK number and CFU-MK. The proportion
of polyploid MK increased greater than fivefold in the presence of TPO. SCF
had little effect on MK ploidy in the presence of TPO, but enhanced ploidy
twofold to threefold in the absence of TPO. IL-3 alone never increased the
level of polyploidization. Rather, it consistently inhibited TPO- and
SCF-induced polyploidization of MK. This inhibition was observed in
cultures with or without SCF or IL-6. Although IL-3 also supported the
proliferation of CD41+ cells and CFU-MK production, the cells that
developed under the influence of IL-3 were phenotypically unusual (CD41dim,
CD42dim) and of relatively low ploidy. Mature MK were not produced. When
added with TPO, IL-3 suppressed polyploidization. Therefore, TPO stimulates
MK growth and maturation, whereas IL-3 stimulates growth without maturation
and may serve to conserve the immature MK compartment.
Volume 89,
Issue 2,
pp. 426-434,
01/15/1997
Copyright © 1997 by The American Society of Hematology
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