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The Mpl-ligand or thrombopoietin or megakaryocyte growth and
differentiative factor has both direct proliferative and differentiative
activities on human megakaryocyte progenitors
N Debili, F Wendling, A Katz, J Guichard, J Breton-Gorius, P Hunt and W Vainchenker
INSERM U 362, Institut Gustave Roussy, Villejuif, France.
Previously, it was believed that megakaryocytopoiesis was regulated by two
types of humoral factors: megakaryocyte colony-stimulating factor (MK-CSF),
which acts on progenitors inducing their proliferation, and thrombopoietin
(TPO), a megakaryocyte(s) (MK) maturational factor that induces platelet
formation. The recently cloned Mpl-ligand (Mpl-L) seems to have both
properties in vivo and in vitro and has also been called TPO. However, it
cannot be excluded that a part of these activities is due to a synergistic
effect with growth factors present in the serum or synthesized by accessory
cells. To delineate the precise TPO (Mpl-L) biologic activities, we
performed serum-free cultures at limiting cell dilution. Target cells were
adult human marrow CD34+CD41+ cells, which represent a highly selected
population of late MK progenitor or transitional cells. Cells were purified
using a flow cytometer equipped with an automatic cloning design unit. We
determined that the recombinant molecule had a biologic activity that
reached a plateau at 10 ng/mL. At this concentration, a linear relationship
between the average MK number per well and the number of cells seeded
(between 1 to 50 cells per well) was observed. At one cell per well, 60% of
the wells contained a single MK at day 5 of culture. Half of these wells
contained only one large MK, whereas the other half contained several MK
(up to 25), demonstrating that TPO has direct proliferative biologic
activity. In contrast, at limiting dilution, none of the other cytokines
tested (stem cell factor [SCF], interleukin- 6 [IL-6], and erythropoietin
[Epo]) were effective, whereas IL-3 showed a mild effect. However, a
combination of SCF plus IL-6 plus IL-3 produced similar results as TPO
alone. Addition of the other cytokines to TPO did not enhance the cloning
efficiency of the CD34+CD41+ cells but increased twofold the average number
of MKs per clone. MKs reached a ploidy of 32N and 64N in the presence of
TPO. The mean ploidy value was approximately 6 and was not modified by
addition of the other cytokines. At the ultrastructural level, a majority
of the MKs showed maturational defects related to an imbalance between the
synthesis of alpha-granules and demarcation membranes. However, a fraction
(about 30%) had a cytoplasmic maturation that exactly mimicked that of
marrow MKs. In addition, proplatelet-shedding MKs were observed in the
cultures, even at limiting dilution. Such a result was not observed with
any other individual cytokines, including the combination of three
cytokines.(ABSTRACT TRUNCATED AT 400 WORDS)
Volume 86,
Issue 7,
pp. 2516-2525,
10/01/1995
Copyright © 1995 by The American Society of Hematology
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