The effect of thrombopoietin on the proliferation and differentiation of murine hematopoietic stem cells

In this study, we explored whether thrombopoietin (Tpo) has a direct in vitro effect on the proliferation and differentiation of long-term repopulating hematopoietic stem cells (LTR-HSC). We previously reported a cell separation method that uses the fluorescence-activated cell sorter selection of low Hoescht 33342/low Rhodamine 123 (low Ho/low Rh) fluorescence cell fractions that are highly enriched for LTRHSC and can reconstitute lethally irradiated recipients with fewer than 20 cells. Low Ho/low Rh cells clone with high proliferative potential in vitro in the presence of stem cell factor (SCF) + interleukin-3 (IL-3) + IL-6 (90% to 100% HPPCFC). Tpo alone did not induce proliferation of these low Ho/low Rh cells. However, in combination with SCF or IL-3, Tpo had several synergistic effects on cell proliferation. When Tpo was added to single growth factors (either SCF or IL-3 or the combination of both), the time required for the first cell division of low Ho/low Rh cells was significantly shortened and their cloning efficiency increased substantially. Moreover, the subsequent clonal expansion at the HE HYPOTHESIS THAT circulating platelet levels and megakaryocytopoiesis are controlled by a humoral regulator was proposed by Yamamotol and named thrombopoietin (Tpo) by Kelemen et a1 in 1958.’ Since that time, many groups have attempted to isolate and purify Tpo. A major step in understanding the regulation of megakaryocytopoiesis was made with the recent identification of the c-rnpl proto~ncogene.”~ Ten years ago, Wendling et alh.7 described a transforming viral complex that induces a myeloproliferative syndrome in mice. The transforming gene v-mpl was subsequently identified by Souryi et a1,8 and the cellular homologue c-mpl was cloned from human HEL cells in 1992.’ Sequence analysis of human3 and murine4,’ c-mpl showed that the encoded protein was likely a novel member of the hematopoietic receptor family. However, its ligand was unknown. Using different strategies, several groups have recently purified the ligand for c-Mpl or have obtained cDNA by functional expression c10ning.~”~ Subsequent characterization of the recombinant protein quickly established that the ligand for c-Mpl is identical to Tpo.””’ Treatment of mice with Tpo substantially increased platelet counts within 5 T