collected platelets were double stained with anti-CD61 and anti-CD42b antibodies, and their number evaluated by a counting bead standard (Figure 4C). of action of eltrombopag when compared to romiplostim, which, as we have shown, induces the proliferation of immature megakaryocytes rather than platelet production, due INSR to the unbalanced activation of AKT and ERK1/2 signaling molecules. In conclusion, our research clarifies the underlying mechanisms that govern the action of eltrombopag on megakaryocyte functions and its relevance in clinical practice. Introduction Hematopoiesis occurs in a complex microenvironment within the bone marrow, which provides an ideal habitat for the production of mature blood cells from the multipotent, self-renewing hematopoietic stem cells (HSCs).1,2 The failure of HSCs to guarantee the physiologic homeostasis of one or more progenitors for circulating blood cells leads to pathologic conditions, such as aplastic anemia (AA) or myelodysplastic syndromes (MDS), characterized by peripheral pancytopenia of heterogeneous severity.3,4 A selective cytopenia of blood platelets, namely Darapladib thrombocytopenia, may occur because of mutations in genes relevant for the functions of maturing megakaryocytic progenitors, as in inherited thrombocytopenias (IT).5 However, thrombocytopenia may also be secondary to viral infections or autoimmune diseases.6,7 It is well known that thrombopoietin, through binding to its receptor (c-Mpl), which is expressed by HSCs and megakaryocytes, is a critical regulator of both HSC homeostasis and megakaryopoiesis.8 The opportunity to synthesize molecules able to mimic the physiologic effect of thrombopoietin on platelet production recently opened new perspectives for the treatment of thrombocytopenic states.9 Among the small, non-peptide c-Mpl agonists,10,11 eltrombopag has been successfully employed to stimulate platelet production in patients suffering from IT caused by mutations of the gene, immune thrombocytopenia (ITP), and thrombocytopenia due to hepatitis C infection.12C14 Moreover, improvement of thrombocytopenia has been obtained in patients Darapladib with acute myeloid leukemia (AML) and MDS.15,16 Interestingly, promising clinical results have also demonstrated multi-lineage responses and the maintenance of normalized hematopoiesis in several patients suffering from AA,17C19 suggesting that eltrombopag may exert a beneficial effect on HSCs recovery by yet unexplored ways. Unfortunately, the use of traditional animal models for studies on the mechanisms of action of eltrombopag is not possible due to its selective activity only in humans and chimpanzees.20 The management of both thrombocytopenias and hematologic malignancies is challenging. Therefore, further improvement of the current knowledge strongly depends on the possibility to create laboratory assays in order to understand the effects of eltrombopag on human Darapladib cells, both in physiologic and pathologic conditions. 21 To this end, we have established a translational study made up of two complementary approaches. The first, based on the development of a culture system for the study of the basic mechanisms of the action of eltrombopag on human HSCs, focuses on the evaluation of the ability to promote megakaryopoiesis and platelet formation. The second, dedicated to the implementation of this knowledge into our recently established silk-based bone marrow model, integrates important physical and physiological elements characterizing the hematopoietic niche, conducive to evaluating platelet production.22 Together our studies Darapladib demonstrate that eltrombopag significantly increases the activation of all the major c-Mpl downstream signaling pathways in a dose-dependent manner, and that this is paralleled by the differentiation of human HSCs, which results in an increased output of mature megakaryocytes which show an improved ability to form proplatelets and release platelets. Furthermore, we propose a novel mechanism explaining such effects on thrombopoiesis through the activation of AKT and ERK1/2 signaling molecules. Methods Cell culture Human cord blood was collected following normal pregnancies and deliveries upon the informed consent Darapladib of the parents; peripheral blood samples were collected from healthy volunteers after written informed consent. All samples were processed in accordance with the ethical committee of the IRCCS Policlinico San Matteo Foundation and the principles of the Declaration of Helsinki. Hematopoietic progenitor cells from cord and peripheral blood were separated by immunomagnetic bead selection, as previously described,23C25 and cultured in StemSpan medium (Stem Cell Technologies, Vancouver, Canada) supplemented with 1% L-glutamine, 1% penicillin-streptomycin, 10 ng/ml interleukin (IL)-6 and IL-11 (PeproTech, London, UK) and 50, 100, 200, 500 or 2000 ng/ml of eltrombopag (kindly supplied by GlaxoSmithKline), at 37C in a 5% CO2 fully humidified atmosphere for 13 days. Instead of eltrombopag, 10 ng/ml recombinant human thrombopoietin (rHuTPO, PeproTech) was always used as the standard.