Expression and function of receptors for extracellular matrix molecules in the differentiation of human megakaryocytes in vitro

The role of adhesive interactions with the extracellular matrix components of the bone marrow (BM) stroma has been widely studied in the differentiation of erythroid and myelomonocytic cells, but not in the mega-karyocytic lineage. The development of efficient culture techniques for the production of megakaryocytes (Mks) from CD34(+) purified BM cells, enables the study of the expression and function of adhesion receptors for collagen (VLA-2). fibronectin (VLA-4 and VLA-5) and laminin (VLA-6) during the maturation of Mks. We have shown that a significant percentage of CFU-MK (roughly 20%) adhere to fibronectin but not to collagen and laminin, The expression and adhesion of Mks developing in liquid culture from BM-CD34(+) cells were tested at days 4, 7 and 10 of incubation. The expression of VLA-2, VLA-5 and VLA-6 on day 10 cultured Mks enabled purification of intermediate and large polyploid Mks by FAGS sorting whereas VLA-4 appeared to label only immature Mks and myeloid cells. We observed that only a small proportion of mature Mks was able to adhere to collagen without spreading at day 10 of culture, whereas 30% of Mks adhered to fibronectin as early as day 4 of incubation, 40% of which also attached to laminin, Our data suggest that VLA-4 may be involved in the adhesion of CFU-MK and immature Mks on fibronectin, then replaced by VLA-5 in the final stages of maturation, The expression of VLA-6 and the number of adherent Mks on laminin increased sharply between day 7 and 10 of incubation. A number of mature polyploid Mks found in day 10 of culture exhibited characteristic features of intense spreading on laminin and fibronectin which were not observed on collagen.