LAUSR

Bone marrow stromal cells provide a proper environment for the development of hematologic lineages. The incorporation of different stromal cells to in vitro culture systems would be an attractive model to study megakaryopoiesis and thrombopoiesis. Our objective was to evaluate the participation of different types of stromal cells on in vitro megakaryopoiesis, thrombopoiesis and megakaryocyte (MK) survival. CD34-positive progenitors from umbilical cord blood were differentiated into MK precursors and then co-cultured with umbilical vein endothelial cells (HUVEC), bone marrow mesenchymal stem cells (MSCs), skin fibroblasts (SF) (all human) or mouse fibroblast cell line (L929). The number of MKs (CD61-positive cells) was increased in the presence of HUVEC and SF while L929 cells decreased total and mature MK count. Concerning thrombopoiesis, HUVEC increased proplatelet (PP)-producing MKs, while MSCs, L929 and SF had the opposite effect (immunofluorescence staining and microscopic analysis).  MK survival was enhanced in MSC and SF co-cultures, as assessed by evaluation of pyknotic nuclei. However, HUVEC and L929 did not prevent apoptosis of MKs. Reciprocally, the cloning efficiency of MSCs was decreased in the presence of MKs, while the ability of stromal cells (either MSCs or SF) to produce the extracellular matrix proteins type III collagen, fibronectin, dermatan sulfate, heparan sulfate and P4HB was preserved. These data indicate that each stromal cell type performs distinctive functions, which differentially modulate MK growth and platelet production, and, at the same time, that MKs also modify stromal cells behavior. Overall, our results highlight the relevance of considering the influence of stromal cells in MK research as well as the close interplay of different cell types within the bone marrow milieu.