Recombinant human c-Mpl ligand (thrombopoietin) not only acts on megakaryocyte progenitors, but also on erythroid and multipotential progenitors in vitro.
To determine the hematopoietic actions of recombinant human c-Mpl ligand (thrombopoietin [TPO]), we studied its effects on the proliferation and differentiation of highly purified CD34+ blood progenitors in plasma-containing and serum-free culture. TPO alone promoted the growth of small megakaryocyte colonies (CFU-Meg) in numbers two to three times greater than those produced by interleukin (IL)-3. The combination of TPO and stem cell factor (SCF) exerted a significant synergistic effect on CFU-Meg formation. In the presence of TPO and IL-3 or granulocyte/macrophage-colony stimulating factor (GM-CSF), a significant number of mixed colonies (CFU-Mix) were observed. The combination of TPO and Epo did not increase the number of CFU-Meg, but did support erythroid-burst (BFU-E) and CFU-Mix colony formation. Interestingly, the combination of TPO with cytokines known to have burst-promoting activity (BPA), including IL-3, GM-CSF, IL-9, and SCF, increased the number of BFU-E and CFU-Mix in the presence of Epo. The BPA of TPO was further investigated by delayed addition of Epo on day 6 after incubation with TPO from day 0. None of the BFU-E or CFU-Mix survived, indicating that TPO acted as a costimulant exclusively for Epo. Moreover, a neutralizing anti-human Mpl receptor polyclonal antibody completely abrogated the BPA of TPO, demonstrating that this effect was mediated through the Mpl receptor. Finally, experiments in single-cell clone sorting and serum-free culture clearly demonstrated that a combination of TPO and Epo directly supported BFU-E and CFU-Mix. These results suggest that TPO acts not only in megakaryocytopoiesis but also in the early stage of hematopoiesis.
Tanimukai
S
S
Second Department of Surgery, Kyoto Prefectural University of Medicine, Kamigyoku, Japan.
Kimura
T
T
Sakabe
H
H
Ohmizono
Y
Y
Kato
T
T
Miyazaki
H
H
Yamagishi
H
H
Sonoda
Y
Y
D018952:Antigens, CD34 / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D015672:Erythroid Precursor Cells / Q000187:drug effects*; D004920:Erythropoiesis / Q000187:drug effects*; D004921:Erythropoietin / Q000494:pharmacology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000008:administration & dosage; D006410:Hematopoiesis / Q000187:drug effects*; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology*; D006801:Humans; D007377:Interleukin-3 / Q000008:administration & dosage; D008297:Male; D008533:Megakaryocytes / Q000166:cytology*; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins / Q000502:physiology; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D013736:Testicular Neoplasms / Q000097:blood; D013926:Thrombopoietin / Q000494:pharmacology*
D018952:Antigens, CD34; D016298:Hematopoietic Cell Growth Factors; D007377:Interleukin-3; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D004921:Erythropoietin; C082218:MPL protein, human; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D013926:Thrombopoietin