Blood platelets play a central role in hemostasis and maintenance of vascular integrity. A severe decreased platelet count (generally <30,000 platelets / μl), frequently observed in patients treated 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, these are targets for the development of immune-related or drug-induced thrombocytopenia, 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 (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 aims to improve our knowledge on platelet biogenesis using mouse animal models. It will be performed in the context of the team’s efforts 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 involved in this very active (reactive) megakaryopoiesis. We anticipate to deduce methods to improve yield in vitro differentiation of MKs.
We will first analyze the in vivo parameters associated with 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 characterized by the end of the treatment of diphtheria toxin. The megakaryocytic commitment of HPCs and multipotent progenitors will be assessed in the population and in vitro culture assays.
During this time, we will check for the development of MKs by flow cytometry. These analyzes will allow us to check the extent of the synchronization of megakaryopoiesis, as suggested by the analysis of bone marrow samples by microscopy on the day of the early platelet count restoration. DMS-expanded mature MKs by in vitro differentiation. The modulation of the gene expression in maturating MKs will be checked by RNA-seq techniques.
Bone marrow mesenchymal cells will be analyzed by cytometry, attention will be focused on CXCL12-abundant reticular (CAR) and Nestin + cells. Gene expression in these cells will also be 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 will be challenged in vitro with MKs and stromal cells.

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