The proposed approach to the megakaryocyte progenitor cell compartment in mouse bone marrow involves the use of fluorescence-activated cell sorting on a multiparameter basis for the isolation and characterization of different progenitor classes using new quantitative cellular, cytochemical and immunofluorescent assays. Progenitors in marrow or cell fractions enriched for progenitors by centrifugal elutriation and percoll density gradient centrifugation, will be labeled with Hoechst 33342 as a supravital DNA stain and heterologous and monoclonal anti-megakaryocyte or antiplatelet antibodies conjugated with FITC for surface immunofluorescence as markers of differentiation and analyzed with a FACS IV. Progenitors will be sorted and assayed in vitro in plasma or agar cultures for clonogenicity (CFU-M). The DNA content, cell size (light scatter) and differentiation (positivity for antibodies or substrates) of individual progenitors will be related to their proliferative (mitotic) activity and the distribution of polyploidy among megakaryocytes in individual colonies formed by CFU-M. These data, together with the use of antimitotic agents and S-phase specific drugs as probes of polyploidization and proliferative activity, will be used to elucidate the interplay of mitotic and endoreduplicative events among different progenitors. The cytokinetic basis for the physiologic regulation of megakaryocyte production will be tested in three experimental mouse models. The recovery of pluripotent stem cells and CFU-M in femoral marrow and changes in their proliferative state as well as the distribution of polyploidy among progenitors obtained by flow cytometry will be quantitated as a function of time in mice following a) the depletion (both acute and chronic) and repopulation of platelet and megakaryocyte subpopulations following antibody-mediated cytolysis; b) the inhibition of thromboxane B2 synthesis in megakaryocytes following aspirin ingestion; and c) hematopoietic reconstitution in the regenerating marrow of lethally-irradiated mice transplanted with normal, syngeneic marrow. These data will elucidate the physiologic importance of various progenitor cell populations and define target cells under regulatory control. They will provide a cellular basis for understanding how the response of mitotic and endoreduplicative events among progenitors results in the production of megakaryocytes in specific ploidy classes.
