Thrombopoietin directly and potently stimulates multilineage growth and progenitor cell expansion from primitive (CD34+ CD38-) human bone marrow progenitor cells: distinct and key interactions with the ligands for c-kit and flt3, and inhibitory effects of TGF-beta and TNF-alpha.
Thrombopoietin (Tpo) is a primary regulator of megakaryocyte and platelet production. However, studies in c-mpl-deficient mice suggest that Tpo might also play an important role in early hemopoiesis. Here, the direct ability of Tpo to stimulate stroma-independent growth, multilineage differentiation, and progenitor cell expansion from single primitive CD34+ CD38- human bone marrow cells was investigated. Tpo alone stimulated limited clonal growth, but synergized with c-kit ligand (KL), flt3 ligand (FL), or IL-3 to potently enhance clonogenic growth. Whereas KL and FL in combination stimulated the clonal growth of only 3% of CD34+ CD38- cells, 40% of CD34+ CD38- cells were recruited by KL+FL+Tpo, demonstrating that Tpo promotes the growth of a high fraction of CD34+ CD38- progenitor cells. Additional cytokines (IL-3, IL-6, and erythropoietin (Epo)) did not significantly enhance clonal growth above that observed in response to KL+FL+Tpo. In contrast, Tpo enhanced clonogenic growth in response to KL+FL+IL-3+IL-6+Epo by as much as 80%, implicating a key role for this cytokine in early hemopoiesis. Importantly, we also demonstrate that the majority of Tpo-recruited CD34+ CD38- progenitor cells have a multilineage differentiation potential, and that Tpo promotes prolonged expansion of multipotent progenitors. Specifically, whereas progenitor cells were reduced in cultures containing only KL+FL, addition of Tpo resulted in 40-fold expansion of multipotent progenitors following a 14-day incubation. Finally, we identified inhibitors of Tpo-induced progenitor cell growth, in that TGF-beta as well as TNF-alpha almost completely abrogated the growth of CD34+ CD38- progenitor cells in response to Tpo alone as well as KL+FL+Tpo.
Ramsfjell
V
V
Hipple Cancer Research Center, Dayton, OH 45439, USA. Veslemoy.Ramsfjell@med.lu.se
Borge
O J
OJ
Cui
L
L
Jacobsen
S E
SE
D000818:Animals; D018952:Antigens, CD34; D001853:Bone Marrow / Q000378:metabolism; D001854:Bone Marrow Cells; D002454:Cell Differentiation / Q000187:drug effects; D019070:Cell Lineage / Q000187:drug effects*; D002478:Cells, Cultured; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects; D006801:Humans; D051379:Mice; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D013926:Thrombopoietin / Q000494:pharmacology*; D016212:Transforming Growth Factor beta / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D051941:fms-Like Tyrosine Kinase 3
D018952:Antigens, CD34; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D014409:Tumor Necrosis Factor-alpha; D013926:Thrombopoietin; C497970:FLT3 protein, human; C497971:Flt3 protein, mouse; D019009:Proto-Oncogene Proteins c-kit; D020794:Receptor Protein-Tyrosine Kinases; D051941:fms-Like Tyrosine Kinase 3