HEMATOPOIESIS IN MICE FETAL SPLEEN (R. Golub)

In all vertebrates, the spleen is an asymmetric organ located next to the stomach which is in direct contact with omemtum and pancreas. The splenic anlagen has been described as a mesenchyme bud in the dorsal mesogastrium. Considered as mesoderm-derived, the early stages of spleen development are difficult to study as no specific marker is known. In adult, the spleen is a secondary lymphoid organ with a complex structure correlated to precise functions during innate and adaptative immune responses. The key steps of the adult splenic architecture are the acquisition of hematopoietic capacities during fetal life, followed by the « regionalization » during the neonatal period.

To study the hematopoietic processes and their implication during the organogenesis, we developed several tools and methodologies. Among others, organ cultures of fetal spleen to develop « in situ » hematopoietic differentiation and reconstitution using congenic conditions. We also characterized stromal cells from E15.5 fetal spleen.

We showed that E13 fetal spleen already contains CSH with identical reconstitution capacities than those isolated from fetal liver. However, in this environment, CSH cannot expand but only differentiate, contrary to fetal liver or newborn bone marrow. We showed that fetal spleen environment is totally devoted to myeloid differentiation. On fetal spleen stroma, all cells cultured for one week are macrophages (F4/80+ Mac1+ phenotype). Moreover, proliferation of myeloid precursors is highly stimulated by fetal spleen stromal cells. In parallel, we analyze what influence the CSH choice between myeloid and lymphoid fates.
Different populations of hematopoietic precursors have been isolated and we studied their differentiation capacity under clonal conditions. They are characterized by CD4 expression and can be subdivided in subsets depending on RAG2 expression levels. We show that these precursors can develop in situ and represent the lymphoid populations found in fetal spleen from E16.

Now, we study the impact of stromal spleen microenvironment on the fetal hematopoiesis and organogenesis. We have identified populations that are known to induce secondary lymphoid organs. Their implication in the architecture of different splenic components is under consideration. We also develop a study on interactions between stroma and hematopoietic cells in patient with splenomegaly syndrome. We are especially interested by the hematopoietic niches during extramedullar metaplasy in myelofibrosis.

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