MEGAKARYOCYTIC DEVELOPMENT IN LIQUID CULTURES OF CRYOPRESERVED LEUKOCYTE STEM CELL CONCENTRATES FROM CHRONIC MYELOGENOUS LEUKEMIA PATIENTS

The proliferation and differentiation of human megakaryocytes in liquid culture has been obtained using cryopreserved light density blood cell concentrates from chronic myelogenous leukemia (CML) patients. These cryopreserved leukocytes concentrates contain a large number of viable granulo-monocytic, erythroid and megakaryocytic committed stem cells. A high number of spontaneous megakaryocytic colonies was observed in semisolid cultures plated with the CML leukocytes concentrates. A liquid culture system using RPMI 1640 supplemented with 20% human plasma (HP) has been defined where maturing megakaryocytes make up 20 to 60% of the total cells after 14 days of incubation. The same cell suspension cultured in medium supplemented with 20% foetal calf serum (FCS) showed poor megakaryocytic cell development. The megakaryocytic nature of the cells produced in HP supplemented cultures was confirmed by cytological studies and indirect immunofluorescence labeling using monoclonal antibodies (MoAb) against membrane platelet GPIb and Ilbllla, and intracellular antigens like fibrinogen and von Willebrand factor.

Ploidy of the cultured cells was studied after labeling with propidium iodide and the DNA fluorescence determined using the fluorescence activated cell sorter (FACSIV). Peaks of 8N, 16N and 32N cells were observed from HP supplemented cultures representing about 20% of the cells reacting with a GP11b111 a MoAb, while very few cells greater than 4N were observed in FCS supplemented cultures. The megakaryocytes produced in HP cultures could be further enriched by cell sorting on the FACSIV after labeling with an anti-IIbIIIa MoAb. Depending on the initial megakaryocytic concentration of the cells cultured, one to 2 é 10⁶ megakaryocytes per hour could be harvested. Thus, cryopreserved CML blood stem cell concentrates seem to offer a reproducible source of human megakaryocytes which retain their capacity to proliferate and differentiate in liquid cultures supplemented with human plasma. These megakaryocytes can be used for the study of platelet glycoprotein biosynthesis as well as the regulation of megakaryocytopoiesis.