THROMBOPOIETIN DIRECTLY AND POTENTLY STIMULATES MULTILINEAGE GROWTH AND PROGENITOR-CELL EXPANSION FROM PRIMITIVE (CD34(-)) 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()CD38()

Thrombopoietin (Tpo) is a primary regulator of megakaryocyte and plate let 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, multilin eage differentiation, and progenitor cell expansion from single primit ive CD34(+)CD38(-) human bone marrow cells was investigated. Tpo alone stimulated limited clonal growth, but synergized with c-kit ligand (K L), flt3 ligand (FL), or IL-3 to potently enhance clonogenic growth. W hereas 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 fra ction of CD34(+)CD38(-) progenitor cells, Additional cytokines (IL-3, IL-6, and erythropoietin (Epo)) did not significantly enhance clonal g rowth above that observed in response to KL(+)FL(+)Tpo. In contrast, T po 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 hemopo iesis, Importantly, we also demonstrate that the majority of Tpo-recru ited CD34(+)CD38(-) progenitor cells have a multilineage differentiati on potential, and that Tpo promotes prolonged expansion of multipotent progenitors. Specifically, whereas progenitor cells were reduced in c ultures containing only KL+FL, addition of Tpo resulted in 40-fold exp ansion of multipotent progenitors following a 14-day incubation. Final ly, we identified inhibitors of Tpo-induced progenitor cell growth, in that TGF-beta as well as TNF-alpha almost completely abrogated the gr owth of CD34(+)CD38(-) progenitor cells in response to Tpo alone as we ll as KL+FL+Tpo.