HoxA2 Regulates Proliferation of an Embryonic Megakaryocyte Progenitor, Which Can Effectively Produce Platelets In Vitro.

Abstract

Megakaryocytes are rare cells in the bone marrow, and are solely responsible for thrombopoiesis throughout the life of the organism. Various transcription factors regulating megakaryopoiesis have been identified, including GATA1, Fli1 and NF-E2, which are important for the maturation and differentiation of megakaryocytes and SCL and FOG1, which play important roles in the specification and proliferation of the early megakaryocyte progenitor but no single factor capable of sustaining long-term proliferation of the megakaryocyte progenitor has been identified. The rarity of this progenitor, and the difficulty of proliferating megakaryocytes in vitro have seriously hampered the effort to produce large quantities platelets in vitro, and to date the only source of platelets for transplantation remains human donors. In this study, through a gain of function screen of Hox genes in which each paralog group was tested, we identified HoxA2 as an important transcription factor for the proliferation of early embryonic megakaryocytic progenitors. HoxA2 promoted the outgrowth of undifferentiated megakaryocyte progenitors when transduced into precirculation yolk sac stem and progenitor cells, however it had no effect on megakaryocytic progenitors derived from definitive HSCs of adult bone marrow. To study this unique embryonic regulatory circuit further, we generated an inducible mouse embryonic stem cell line, in which the expression of murine HoxA2 is regulated by doxycycline. Embryoid body differentiation of these cells, followed by sorting and reculture of the Kit+/CD41+ hematopoietic progenitor fraction at day 6 in the presence of continual HoxA2 expression allowed undifferentiated c-Kit+/CD41+/CD45+ megakaryocyte progenitors to proliferate for upwards of 2 months. When doxycycline is removed, numerous gene expression changes take plase, and these progenitors differentiate into mature c-Kitneg/CD41high/CD45neg megakaryocytes, which undergo proplatelet formation, releasing large quantities of platelets (∼107/ml) into the medium. As this rate of production can be sustained for weeks, a highly efficient platelet bioreactor is enabled. The platelets express the integrin αIIb-β3 (GPIIb-IIIa complex), and upon stimulation they bind fibrinogen, demonstrating functionality. We are currently investigating the transplantation potential of these cells.Our data suggest that adult and embryonic megakaryopoiesis have distinct regulatory circuits, and demonstrate a novel and uniquely embryonic role for HoxA2, which opens up the possibility of producing industrial quantities of platelets in vitro for therapeutic purposes.