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

Thrombopoietin (Tpo), the ligand for the c-mpl receptor, is a major re gulator of platelet production in vivo. Treatment of mice with purifie d recombinant Tpo increases platelet count fourfold and expands colony -forming unit-megakaryocyte (CFU-Meg) numbers. Other cytokines includi ng interleukin-3 (IL-3), IL-6, IL-11, erythropoietin (Epo), and stem c ell factor (SCF) can stimulate megakaryopoiesis. Therefore, we examine d the effects of recombinant murine Tpo in combination with these cyto kines on megakaryopoiesis in vitro. Murine marrow cells were cultured in agar in Iscove's modified Dulbecco's medium (IMDM) supplemented wit h 10% horse serum and beta-mercaptoethanol in the presence of recombin ant growth factors, and CFU-Meg colonies were counted on day 5. Megaka ryocyte ploidy was analyzed using murine marrow cells cultured for 5 d ays in IMDM supplemented with 1% nutridoma-SP and recombinant growth f actors. Megakaryocytes were identified by labeling with the 4A5 antibo dy 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 mode st effect on CFU-Meg growth, the combinations of SCF plus Tpo, Epo plu s 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 a ppeared to be approximately additive. Fifty percent of megakaryocytes generated in cultures containing IL-3 or Epo displayed less than or eq ual to 16N ploidy. In contrast, cultures containing Tpo uniquely gener ated large numbers (30% to 35% of the total) of megakaryocytes with gr eater than or equal to 64N ploidy. These results show that Tpo stimula tes both proliferation of committed megakaryocytic progenitor cells an d maturation of megakaryocytes, and that two multipotent cytokines, SC F and IL-11, as well as a late-acting erythroid cytokine, Epo, can syn ergize with Tpo to stimulate proliferation of CFU-Meg. (C) 1995 by The American Society of Hematology.