Thrombopoietin, the ligand for the Mpl receptor, synergizes with steel factor and other early acting cytokines in supporting proliferation of primitive hematopoietic progenitors of mice

Abstract

Recently, the ligand for the Mpl receptor (ML) was identified to be thrombopoietin, the principal regulator of megakaryocytopoiesis and thrombopoiesis. We examined the effects of ML, as a single factor or in combinations with early acting factors such as steel factor (SF), interleukin (IL)-3, IL-1, IL-6, and granulocyte colony-stimulating factor (G-CSF), on colony formation from primitive progenitors of mice. Cells enriched for cell cycle dormant primitive progenitors were isolated from bone marrow cells of 5-fluorouracil (5-FU)-treated mice by a combination of Nycodenz density gradient separation, immunomagnetic selection for lineage-negative cells, and fluorescence- activated cell sorter (FACS) sorting for Ly-6A/E+Kit+ cells. ML, in the presence of erythropoietin, could support the formation of only a few megakaryocyte colonies. However, ML acted synergistically with SF or IL- 3 to support the formation of multiple types of hematopoietic colonies including multilineage colonies. Effects of the combination of ML and SF on multipotential progenitors were not mediated through other cells, as demonstrated by micromanipulation of individual progenitors. In suspension culture, the combination of ML and SF increased the number of multipotential progenitors. ML also acted synergistically with IL- 11, IL-6, or G-CSF to support colony formation in serum-containing, but not in serum-free, cultures. However, the multilineage colony formation seen in serum-containing culture was completely abrogated by addition of ACK2, a neutralizing antibody to Kit protein. Serial observation (mapping studies) of colony development from multipotential progenitors suggested that ML triggers the cell division of dormant progenitors. Based on these observations, we propose that ML can function as an early acting cytokine and stimulate the proliferation of cell cycle dormant progenitors by shortening their G0 period.

Effects of recombinant thrombopoietin on the growth of murine primitive and committed hematopoietic progenitors in serum-free culture

“…Stem cell factor has been proposed to be a candidate for these undefined factors. 15 Our data of the cytokine stimulation on murine megakaryocyte progenitors in serum-free culture are in agreement with previous studies using isolated human CD34 + cells. [12][13][14] Among the cytokines tested, only rmIL-3 or rmTpo supported the growth of CFU-Mk when added to…”

“…employed for investigating the action of Tpo on human, but not murine, megakaryocytic progenitors and on primitive hematopoietic cells of humans and mice. [12][13][14][15][16] Experimental data from the murine system, in comparison with those of human system, should be valuable for developing the basic in vitro studies to in vivo models or preclinical studies. The present study evaluates the effect of Tpo in combination with other cytokines on the growth of megakaryocytic progenitors (CFU-Mk), granulocyte-macrophage progenitors (CFU-granulocyte-macrophage (GM)), erythroid progenitors (burst-forming unit (BFU)-erythroid (E)) in normal murine marrow, and of primitive progenitors (high-proliferative potential colony-forming cells; (HPP-CFC)) from 5-fluorouracil (5FU)-treated marrow in the serum-free clonogenic assay system.…”

Tyrosine-599 of the c-Mpl receptor is required for Shc phosphorylation and the induction of cellular differentiation.

“…Deficiencies in progenitor cells of other haemopoietic lineages in mp'lmice (Alexander et al, 1996) also suggest a role for TPO in the production of immature haemopoietic cells. Indeed, recent studies in vitro support a direct role for TPO in supporting proliferation of primitive haemopoietic progenitors (Ku et al, 1996).…”

Development of a liquid culture system for megakaryocyte terminal differentiation: fibrinogen promotes megakaryocytopoiesis but not thrombopoiesis

“…Thrombopoietin (TPO) plays a major role in forming megakaryocytes and producing platelet 4 (PF4) (Caen et al, 1999). Stem cell factor (SCF), interleukin 3 (IL-3), IL-6, IL-11 and basic fibroblast growth factor (bFGF) also contribute to megakaryocyte formation (Broudy et al, 1995;Ku et al, 1996). The activating signal transducers and activators 3 and 5 (STAT-3 and STAT)5) are thought to contribute to the activation of the TPO receptor (Drachman et al, 1997).…”