We demonstrated that the simple combination of two growth factors (the ligand for fK3, FL and the Bgand for c-mpl or thrombopoietin, TPO) allowed, in vitro, extensive amplification and self-renewal of very primitive megakaryocyte progenitors from cord blood (Piacibello, Blood 1997, Leukemia, in press). This phenomenom could be attributable, at least in part, to the unique properties of CB hematopoietic progenitors, which are known to possess higher proliferative potentials than the corresponding adult progenitors. Bone marrow is another rich source of progenitors belonging to all of the hematopoietic lineages. In an attempt to further evaluate the capacity of FL and TPO, alone or in combination with other growth factors (IL3, IL6, and SCF) to act on very primitive bone marrow megakaryocitic cells, we set up stroma-free long term suspension cultures. Twenty thousand higly purified CD34 BM cells were cultured in liquid culture for several weeks in the presence of FL(50 ng/mL), TPO (10 ng/mL), IL3 (10 ng/mL), IL6 (10 ng/mL), and SCF (50 ng/mL), which were added alone or in various combinations at the beginning of the cultures and then replaced twice a week. Every week the cultures were demidapopulated by removal of one half the culture volume, which was replaced with fresh medium and/or growth factors. Cells of the harvested media were assayed weekly for CFU-Mk progenitors. After 2 weeks of liquid cultures among the single factors tested, IL3 was the most potent stimulator of CFU-Mk output (1.5-fold the input number); FL or TPO alone were incapable of sustaining CFU-Mk output; the combination of IL3, FL, TPO and SCF allowed a better expansion (8-fold the input number). Analisis of the time course of CFU-Mk expansion showed that the expansion of US-supplemented culture was only transient: indeed at the end of the 8 weeks of liquid culture CFU-Mk were no longer detectable.The expansion of CFU-Mk during the initial weeks of culture, in the presence of FL+TPO(±SCF±IL6), was not impressive; however, by week 5 it became more relevant and at week 12 it was 40-fold the input number. CFU-Mk were no longer detectable by week 14. Our data show that the expansion of more primitive Mk progenitors from bone marrow is certainly less impressive and prolonged than that of the corresponding CB progenitors.
|Number of pages||1|
|Publication status||Published - 1998|
ASJC Scopus subject areas
- Cancer Research
- Cell Biology