The long term goal of this research project is to acquire a comprehensive understanding of the regulation of human megakaryocytopoiesis. The general hypothesis to be tested is that megakaryocytopoiesis is subject to both immune cell mediated and autoregulatory influences whose effects may be detectable at the molecular level.
Three specific aims are formulated to test this hypothesis.
Aim #1 will be to determine the importance of ancillary bone marrow cells (T lymphocytes, monocyte- macrophages, and natural killer cells) in regulating growth and development of human megakaryocyte progenitor cells (CFU- Meg), in vitro and in vivo.
Aim #2 will be to examine the effect of mature megakaryocytes and platelets, or their constituents/products, on the growth of CFU-MEG in culture. Inhibition of growth would imply the existance of an autoregulatory circuit for controlling cell proliferation in this compartment. We will then determine if such effects are exherted directly on CFU-MEG or via interaction with the ancillary marrow cell populations just enumerated.
Aim #3 will be to examine the expression of proto-oncogenes, such as c-myb, p53, c-fes,and c-fms, in megakaryocytes obtained from normal human bone marrow, and from patients with disorders that are accompanied by megakaryocyte hyperplasias. In addition megakaryocytes at different maturation stages will be examined, and development of cell size, ploidy, and apparent maturation level will be correlated with the expression of the genes listed above The core methodologies for this project will be 1) hematopoietic cell culture in a variety of semi-solid support matricies including plasma clot and methylcellulose, 2) counterflow centrigual elutriation for the isolation and enrichment of human bone marrow megakaryocytes, and 3) in situ hybridization of cells with tritiated or biotinylated cDNA for detection of proto-oncogene expression in cells obtained by elutriation, and in cells grown in vitro from their earliest progenitor cell, or colony forming unit. This study will also make extensive use of monoclonal antibodies for purification of ancillary marrow effector cells, and hematopoietic progenitor cells. The antibodies will be employed for cell identification, and in concert with immunoadsorption, or flow cytometry for cell purifications. It is well established that platelets are involved in a number of bodily processes, both homestatic and pathologic in nature. An example of the former is the absolute requirement of platelets for normal hemostasis, and examples of the latter include the involvement of platelets in the metastasis of tumor cells, and in atherogenesis. Since the functional properties of platelets are pre-determined by their cell of origin, i.e. the megakaryocyte, it seems clear that a better understanding of the biology of these cells will be both scientifically and clinically rewarding.
| Wang, D L; Annamalai, A E; Ghosh, S et al. (1990) Human platelet factor V is crosslinked to actin by FXIIIa during platelet activation by thrombin. Thromb Res 57:39-57 |
