Blood platelets play a vital role in stopping bleeding. Patients on chemotherapy, polytrauma or with quantitative or qualitative abnormalities of platelets, are at risk of hemorrhagic accidents sometimes serious, requiring platelet transfusions. Platelets are produced by megakaryocytes, themselves differentiated from hematopoietic stem cells within the bone marrow, a complex medium where stromal and hematopoietic cells coexist, as well as the extracellular matrix. During the maturation phase, the megakaryocytes elaborate a complex network of intracellular membranes which will become the membranes of the proplaquettes, cytoplasmic extensions from which the platelets are released. Because of its structure, the bone marrow behaves like a stiff, highly dynamic tissue, with an uninterrupted flow of cells that migrate to the sinusoidal vessels to migrate into the bloodstream. The contacts between the cells and the adhesion to the extracellular matrix generate local physical constraints which necessarily affect the cells in differentiation. Our previous observations in 3D culture have shown that megakaryocytes have a different morphology (modification of the intracellular membrane network) and a capacity to form platelets variable depending on the rigidity of the surrounding environment.

Thesis project: We will tackle the questions of knowing:
1) if modification of the morphology of megakaryocytes is a prerequisite for the formation of propellants;
2) what is the mechanotransduction responsible for remodeling these internal membranes;
3) what are the extracellular signals that trigger this remodeling and thus promote the initiation of propellants.
We will study i) the morphology of megakaryocytes during the release of proplatelets in vivo and ex vivo; ii) the impact of the 3D culture in hydrogels of known rigidity, functionalized or not covalently with proteins of the matrix. Such gels are commercially available; iii) the in vivo importance of extracellular matrix adhesion in the stroma or sinusoidal vessels, using mouse models whose megakaryocytes are devoid of integrins, the main protein receptors in the matrix. These mice are available in the laboratory.

Technical approaches: This study will involve: (i) isolation and culture techniques for murine hematopoietic stem cells; ii) immunofluorescence techniques on cells and tissues and observation by confocal microscopy; iii) the use of electron microscopy to visualize the ultrastructure of differentiated megakaryocytes in vitro or within the marrow of mice.

Expected results: The realization of this work will on the one hand better understand the mechanisms involved in the production of blood platelets in vivo, and on the other hand will highlight important determinants to improve the production of platelets in vitro from stem cells, genetically manipulated or not.

Read more