Platelets are essential elements of hemostasis. Certain pathologies or treatments lead to an alteration of the number or functions of the platelets. The transfusion of platelets from the blood donation is then a preferred treatment to ensure the control of hemostasis. The growing need for donations associated with the short shelf life of wafers often leads to situations of tense flows and heavy pressure on logistics networks. It is therefore useful to consider the production of platelets in vitro. This type of production is limited to the research laboratory and its efficiency is still very insufficient to consider large-scale production. The goal of this project is to better understand the mechanisms of platelet production in order to improve their in vitro production. Platelets are produced by specialized cells of the bone marrow, the megakaryocytes (MK). Hematopoietic stem cells (HSCs) are the source of all the cells of hematopoietic lineages, in particular MKs. Megakaryopoiesis is the complex process that leads to the development of MK from HSCs. HSCs produce hematopoietic progenitors that progressively switch to the megakaryocytic (MKp) pathway through proliferation, differentiation and maturation phases leading to the production of mature MKs capable of releasing into the bloodstream the 1011 platelets produced. per day in humans. For all the steps that go from CSH to mature MK releasing platelets into the circulation, the hematopoietic cells are in contact with a particular microenvironment that participates in the control and regulation of each step. The microenvironment consists of cellular elements (stromal cells, endothelial cells), non-cellular elements (extracellular matrix, cytokines and growth factors, calcium and oxygen). During megakaryopoiesis, a succession of microenvironments will accompany the expansion of MK progenitors and their differentiation and maturation. The fetal liver is a transient site for the production of blood cells prior to the establishment of definitive hematopoiesis in the bone marrow. Preliminary results obtained in our laboratory showed that the fetal liver included several populations of stromal cells, one of which favors the production of MK from CSH. The other populations tend to repress the commitment of the HSCs to the megakaryocytic pathway but seem however able to support the differentiation and maturation of progenitors already committed to the megakaryocytic pathway.
The aim of this project is therefore to understand how the fetal liver environment can influence the involvement of HSCs in the megakaryocytic pathway and how the microenvironment can be used to improve in vitro platelet production. . We will evaluate in vitro the role of the different cellular elements of the microenvironment in the expansion and differentiation of megakaryocytes. This work will also lead to a reassessment of the hematopoietic hierarchy during megakaryopoiesis. The hematopoietic, stromal and endothelial cells will be sorted by flow cytometry and cultured in several co-culture models established in the laboratory to evaluate the respective roles of each cell type present in the hematopoietic tissues during development. We will determine the mode of action of stromal cells (especially the secretion of particular factors, action by cell-cell contact). The molecular analysis (transcriptome / proteome) of these effector cells in these models will allow us to identify the secreted proteins or cell surface which are the functional elements of the megakaryocytic microenvironment.

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