In Vitro Megakaryocyte Differentiation and Proplatelet Formation in Ph-Negative Classical Myeloproliferative Neoplasms: Distinct Patterns in the Different Clinical Phenotypes

Alessandra Balduini,Annalisa De Silvestri,Stefania Badalucco,Giovanni Barosi,Denis Baev,Maria Teresa Pugliano,Marco Cattaneo,Vittorio Rosti,Christian Schulz

doi:10.1371/journal.pone.0021015

Abstract

BackgroundPh-negative myeloproliferative neoplasms (MPNs) are clonal disorders that include primary myelofibrosis (PMF), polycythemia vera (PV) and essential thrombocythemia (ET). Although the pathogenesis of MPNs is still incompletely understood, an involvement of the megakaryocyte lineage is a distinctive feature.Methodology/Principal FindingsWe analyzed the in vitro megakaryocyte differentiation and proplatelet formation in 30 PMF, 8 ET, 8 PV patients, and 17 healthy controls (CTRL). Megakaryocytes were differentiated from peripheral blood CD34+ or CD45+ cells in the presence of thrombopoietin. Megakaryocyte output was higher in MPN patients than in CTRL with no correlation with the JAK2 V617F mutation. PMF-derived megakaryocytes displayed nuclei with a bulbous appearance, were smaller than ET- or PV-derived megakaryocytes and formed proplatelets that presented several structural alterations. In contrast, ET- and PV-derived megakaryocytes produced more proplatelets with a striking increase in bifurcations and tips compared to both control and PMF. Proplatelets formation was correlated with platelet counts in patient peripheral blood. Patients with pre-fibrotic PMF had a pattern of megakaryocyte proliferation and proplatelet formation that was similar to that of fibrotic PMF and different from that of ET.Conclusions/SignificanceIn conclusion, MPNs are associated with high megakaryocyte proliferative potential. Profound differences in megakaryocyte morphology and proplatelet formation distinguish PMF, both fibrotic and prefibrotic, from ET and PV.

Highlights

Megakaryocytes and platelets, which are their progeny, are highly specialized cells that participate in hemostatic and inflammatory functions
Recent studies pointed to a key role of abnormal megakaryocytopoiesis in the pathogenesis of myeloproliferative neoplasms (MPNs) [12],[13], little is known about the latter stage of megakaryocyte development and proplatelet formation in these diseases
We found that each MPN category displayed peculiar alterations of megakaryocyte differentiation and function in vitro, suggesting that, besides the potential deregulation of bone marrow microenvironment, intrinsic defects of megakaryocyte function contribute to the pathogenesis of MPNs

Summary

Introduction

Megakaryocytes and platelets, which are their progeny, are highly specialized cells that participate in hemostatic and inflammatory functions. Available information on mutations of genes encoding tyrosine kinases and their pathways do not explain entirely the molecular pathogenesis of MPNs and this lack of information contributes to the slow development of effective treatments This justifies the continuous search for new cellular and molecular aberrations that characterize these disorders and could become targets of new therapies. Aberrant proplatelet formation has been shown in bone marrow from patients with MPNs [13] Overall, these data suggest that abnormal megakaryopoiesis is a key feature of MPNs in general and of PMF primarily. These data suggest that abnormal megakaryopoiesis is a key feature of MPNs in general and of PMF primarily It is unknown whether the pathological mechanisms underlying MPNs are caused by intrinsic defects of megakaryocyte function or by abnormalities of the bone marrow microenvironment, which regulates megakaryocyte formation and function. The pathogenesis of MPNs is still incompletely understood, an involvement of the megakaryocyte lineage is a distinctive feature

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Conclusion