Megakaryocytopoiesis in vitro: from the stem cells' perspective

Megakaryocytopoiesis is a complex network regulated by different megakaryocyte (MK)-stimulating factors (i.e., thrombopoietin [TPO], stem cell factor [SCF], interleukin 3 [IL-3], IL-6, IL-11 and GM-CSF). Although all of these factors can affect human and murine megakaryocytopoiesis at different levels of MK development, the effect on very primitive hematopoietic stem cells (HSC) is not well understood. We have further characterized the in vitro biological activity of recombinant murine TPO, SCF and IL-3 on the maturation and proliferation of MK progenitors from different murine primitive hematopoietic cells in a fibrin clot system under serum-free conditions. Neither TPO nor SCF alone induced MK colony formation (CFU-MK) from Lin- Sca+ cells. However, isolated large and mature MKs were observed in the presence of TPO. In contrast, IL-3 exerted a potent effect on CFU-MK formation from Lin- Sca+ cells. On this population of HSC, a significant increase of large MK colonies with mature MK were obtained under those conditions in which TPO was combined with IL-3 or SCF plus IL-3. Similar results were obtained with murine bone marrow cells enriched by primitive progenitors from day 3 post-5-fluorouracil treated mice (5-FUBMC). In contrast, TPO-sensitive precursors were detected in fetal liver cells (FLC). These cells differentiate and proliferate to MK progenitors in the presence of TPO. A significant increase in the number of CFU-MK was induced when TPO was combined with either IL-3 or SCF. On these populations of primitive hematopoietic progenitors, IL-3 induced both the proliferation and differentiation of MK progenitors. Because erythropoietin and TPO share similarities between their molecules and their receptors, we studied whether these growth factors may modulate megakaryocytopoiesis from FLC. Flow cytometry analysis of FLC expressing erythroid markers demonstrated that these cells expressed c-Mpl receptor. In our in vitro studies, although EPO by itself did not induce MK colonies from FLC, it enhanced the proliferative activity of TPO. High ploidy and proplatelet-shedding MK were observed in Lin- Sca+ cells, 5-FUBMC and FLC stimulated with TPO alone or in combination with other MK-stimulating factors. Based on these observations, we propose that TPO, IL-3 and SCF constitute early MK-acting factors with differential proliferative and differentiative activities on murine stem cells. TPO by itself does not appear to be involved in the proliferation of MK progenitors from bone marrow HSC. TPO appears to induce in these cells the commitment toward MK differentiation. However, this growth factor may enhance the proliferative activity of IL-3. IL-3 is an early MK-stimulating factor able to induce in vitro the proliferation and differentiation of MK progenitors from HSC.