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 compon ents of the bone marrow (BM) stroma has been widely studied in the dif ferentiation of erythroid and myelomonocytic cells, but not in the meg a-karyocytic lineage. The development of efficient culture techniques for the production of megakaryocytes (Mks) from CD34(+) purified BM ce lls, enables the study of the expression and function of adhesion rece ptors 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 c ollagen and laminin, The expression and adhesion of Mks developing in liquid culture from BM-CD34(+) cells were tested at days 4, 7 and 10 o f incubation. The expression of VLA-2, VLA-5 and VLA-6 on day 10 cultu red Mks enabled purification of intermediate and large polyploid Mks b y FAGS sorting whereas VLA-4 appeared to label only immature Mks and m yeloid cells. We observed that only a small proportion of mature Mks w as 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 incubat ion, 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 fibron ectin, then replaced by VLA-5 in the final stages of maturation, The e xpression of VLA-6 and the number of adherent Mks on laminin increased sharply between day 7 and 10 of incubation. A number of mature polypl oid Mks found in day 10 of culture exhibited characteristic features o f intense spreading on laminin and fibronectin which were not observed on collagen.