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Thrombopoietin (c-mpl ligand) acts synergistically with erythropoietin,
stem cell factor, and interleukin-11 to enhance murine megakaryocyte colony
growth and increases megakaryocyte ploidy in vitro
VC Broudy, NL Lin and K Kaushansky
University of Washington, Division of Hematology, Seattle, 98195, USA.
Thrombopoietin (Tpo), the ligand for the c-mpl receptor, is a major
regulator of platelet production in vivo. Treatment of mice with purified
recombinant Tpo increases platelet count fourfold and expands
colony-forming unit-megakaryocyte (CFU-Meg) numbers. Other cytokines
including interleukin-3 (IL-3), IL-6, IL-11, erythropoietin (Epo), and stem
cell factor (SCF) can stimulate megakaryopoiesis. Therefore, we examined
the effects of recombinant murine Tpo in combination with these cytokines
on megakaryopoiesis in vitro. Murine marrow cells were cultured in agar in
Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% horse
serum and beta-mercaptoethanol in the presence of recombinant growth
factors, and CFU-Meg colonies were counted on day 5. Megakaryocyte ploidy
was analyzed using murine marrow cells cultured for 5 days in IMDM
supplemented with 1% nutridoma-SP and recombinant growth factors.
Megakaryocytes were identified by labeling with the 4A5 antibody and ploidy
was analyzed by flow cytometry. Tpo supported the growth of CFU-Meg in a
dose-dependent manner. Although the addition of SCF (50 ng/mL), Epo (2
U/mL), or IL-11 (50 ng/mL) alone exerted only a modest effect on CFU-Meg
growth, the combination of SCF plus Tpo, Epo plus Tpo, or IL-11 plus Tpo
resulted in a synergistic enhancement of the number of CFU-Meg colonies.
IL-3 alone supported CFU- Meg colony growth, and the effects of IL-3 plus
Tpo or IL-6 plus Tpo on colony growth appeared to be approximately
additive. Fifty percent of megakaryocytes generated in cultures containing
IL-3 or Epo displayed < or = 16 N ploidy. In contrast, cultures
containing Tpo uniquely generated large numbers (30% to 35% of the total)
of megakaryocytes with > or = 64N ploidy. These results show that Tpo
stimulates both proliferation of committed megakaryocytic progenitor cells
and maturation of megakaryocytes, and that two multipotent cytokines, SCF
and IL-11, as well as a late-acting erythroid cytokine, Epo, can synergize
with Tpo to stimulate proliferation of CFU-Meg.
Volume 85,
Issue 7,
pp. 1719-1726,
04/01/1995
Copyright © 1995 by The American Society of Hematology
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