Regulation of megakaryocytopoiesis by thrombopoietin.

It is clear that thrombopoietin is a major hormonal regulator of megakaryocytopoiesis both in vitro and in vivo, and, thus, blood platelet production. Existing data show that the action, chemical nature, and immunologic properties of thrombopoietin from HEK cell culture medium and either endogenously produced or exogenously administered thrombopoietin from animal sources are similar, if not identical. Absolute identity, however, will require comparisons of amino acid sequences of the two preparations. Recent studies have shown that not only does TSF potentiate the action of meg-CSF, but it also has a direct effect on precursor cells to increase the number of megakaryocytic colonies. Other in vitro work showed that TSF stimulates directly the SAChE+ precursor cells to become mature megakaryocytes and causes FMLC to differentiate into megakaryocytic colonies. In vivo, TSF increases megakaryocyte size and number, it causes an elevation in the number of the SAChE+ precursor cells in mouse marrow and increases the maturation of megakaryocytes. Moreover, TSF increases the endomitosis of megakaryocytes in the marrow of mice, along with elevating the number of megakaryocytic colonies in spleens of lethally irradiated bone marrow reconstituted mice. Platelet production is also stimulated in mice by TSF as evidenced by elevated isotopic incorporation into platelets; it increases platelet sizes, and when administered in high doses TSF elevates platelet counts. Full development of colonies of megakaryocytes may depend on two growth factors. It has been hypothesized that one factor, meg-CSF, is effective in clonal expansion whereas a second factor is predominately involved in the endomitotic phase of megakaryocyte development. Multifactoral regulation has been observed for the other cell lineages, and a general proposal for hematopoietic development has been outlined by Iscove. In this scheme, specificity of erythropoietin to erythroid cell lineage is indicated. Previous work, however, shows that recombinant erythropoietin can act as a meg-CSF stimulus, indicating that much is yet to be learned about the action of hematopoietic regulatory factors. Although the present study showed that TSF may in some circumstances stimulate an early cell in the megakaryocytic series, its major effect is probably on the more differentiated population, leading to maturation of megakaryocytes and platelet production.(ABSTRACT TRUNCATED AT 400 WORDS)

Multivectorial analysis of megakaryocytic differentiation.

The definition of the cytokinetics of megakaryocytic differentiation has rested mainly on morphologic classifications that lack a quantitative data base. The approach that we have developed comprises the systematic combination of new techniques for quantitative megakaryocyte separation and correlated, multiparameter analysis of multiply labeled single cells by flow cytometry. We have coined this approach a multivectorial analysis of megakaryocytic differentiation. Modifications of combined centrifugal elutriation (CE) and density gradient centrifugation permit the rapid collection of large numbers of intact, essentially homogeneous, marrow megakaryocytes. These cells, labeled simultaneously with multiple markers of DNA content and surface membrane immunofluorescence have been subjected to correlated, dualparameter analysis by flow cytometry and cell sorting, in an attempt to relate polyploidization (as a unique megakaryocytic property) to cytoplasmic progress towards end-state platelet formation. This approach provides a reasonable data base for an objective appraisal of the interactive roles of nuclear endoreduplication and cytoplasmic development during differentiation. Furthermore, the potential usefulness of matrix models and other innovations inherent in their exploitation hold the promise of describing regulatory mechanisms that are poorly understood, both in normal megakaryocytes and those produced in disease.

[Proliferation of megakaryocytic precursor cells (CFU-M) in a micro-agar culture system]. / Proliferation von megakaryozytären Vorläuferzellen (CFU-M) in einem Mikroagarkultursystem.

The recently described micro-agar culture system for cloning erythropoietic progenitor cells was used to study the optimum conditions for the growth of CFU-M. In this system human mononuclear cells from normal human bone marrow were suspended in agar and incubated for 12 days. Various concentrations of phytohaemagglutinin lymphocyte conditioned medium (PHA-LCM) and prostaglandin E (PGE) were added to the liquid overlayer in the presence of 2-mercaptoethanol (2-ME) for the stimulation of CFU-M. Human AB serum was used instead of fetal calf serum (FCS) in all experiments. A sigmoidal dose-response curve, with a plateau at a concentration of 5 to 10%, was obtained by the addition of different concentrations of PHA-LCM in the presence of 10(-6)PG-E. Under optimal conditions (5% PHA-LCM, PGE 10(-6)M) a linear relation was obtained between the number of seeded cells and the megakaryocytic colonies formed. For routine morphological analysis the whole agar layer was stained using the Pappenheim method. For further characterization of CFU-M, an immunofluorescence test with rabbit antihuman factor VIII related antigen was performed on the whole agar layer.