Blood platelets play a central role in hemostasis and the maintenance of vascular integrity. A severe decreased platelet count (generally <30 000 platelets/µl), frequently observed in patients subjected to medical treatment (medications, radiation treatment or organ transplant surgery) is associated with high bleeding risks threatening their lives. Platelet transfusion is required in these cases. Today, due to their continuous expansion, major public health issues are addressed by the groups of individuals under anti-cancer therapy and of older adults ─ these latters display a higher risk to develop immune-related or drug-induced thrombocytemia or, bone marrow failure. Thus, the in vitro production of safe blood platelets is a challenging issue for transfusion in a general context where the demand for controlled blood products is sustained. In vivo, the formation of platelets is the result of a complex series of cellular processes which can be divided into two major consecutive series of cellular processes, (i) megakaryopoiesis, which is development of mature megakaryocytes (MK) from bone marrow hematopoietic progenitors and (ii), thrombopoiesis, which is the generation of platelets from mature MKs.
The project intends to improve our knowledge on platelet biogenesis using mouse animal models. It will be performed in the context of team attempting to develop efficient methods of in vitro production of human platelets.

Administration of diphtheria toxin in mice expressing its receptor in MKs for 4 days boost megakaryopoiesis. In addition, under these conditions, bone marrow MKs are hypertrophied. We here propose to characterize the features associated to this dynamic state in order to point to biological pathways involved in this very active (reactive) megakaryopoiesis. We anticipate to deduce methods to improve the yield in vitro differentiation of MKs.
We will first analyze the in vivo parameters associated to experimental reactive megakaryopoiesis, concentrating on the co-integration of MK precursors and stromal in megakaryopoiesis. By flow cytometry, hematopoietic populations, in terms of phenotypes and numbers, will be first characterized at different days after the end of the treatment with diphtheria toxin. The megakaryocytic commitment of HPCs and multipotent progenitors will be assessed on sorted cell population and in vitro culture assays.
During this time course, we will check for the development of MKs by flow cytometry. These analyses will allow to check the extent of the synchronization of megakaryopoiesis, as suggested by the analysis of bone marrow samples by electron microscopy on the day of beginning platelet count restoration. We will investigate whether the committed megakaryocytic cells display an intrinsic advantage to generate DMS-enlarged mature MKs by in vitro differentiation. The modulation of the gene expression in maturating MKs will be checked by RNA-seq techniques, using isolated bone marrow MKs at different times of the response.
Bone marrow mesenchymal cells will be also analyzed by flow cytometry, attention will be focused on CXCL12-abundant reticular (CAR) and Nestin+ cells. Gene expression in these cells will be also checked.
In addition, we propose to use protein arrays to define how reactive megakaryopoiesis changes the profile of soluble proteins released by bone marrow cells.
The biological relevance of these observations on mouse cells will be challenged in in vitro assays with human MKs and stromal cells.

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