EFFECTS OF CYTOKINES ON PLATELET PRODUCTION FROM BLOOD AND MARROW CD34(+) CELLS

The late stages of megakaryocytopoiesis, consisting of the terminal pr ocesses of cytoplasmic maturation and platelet shedding, remain poorly understood. A simple liquid culture system using CD34(+) cells in ser um-free medium has been developed to study the regulation of platelet production in vitro. Platelets produced in vitro were enumerated by fl ow cytometry. A truncated form of human Mpl-Ligand conjugated to polye thylene glycol (PEG-rHuMGDF) played a crucial role in both proplatelet formation and platelet production. A combination of stem cell factor (SCF), interleukin-3 (IL-3), and IL-6 was as potent as PEG-rHuMGDF for the growth of megakaryocytes (MKs). However, the number of proplatele t-displaying MKs and platelets was increased 10-fold when PEG-rHuMGDF was used. Peripheral blood mobilized CD34(+) cells gave rise to a thre efold augmentation of platelets compared with marrow CD34(+) cells. Th is finding was related to the higher proliferative capacity of the for mer population because the proportion of proplatelet-displaying MKs wa s similar for both types of CD34(+) cells. The production of platelets per MK from CD34(+) cells was low, perhaps because of the low ploidy of the cultured MKs. This defect in polyploidization correlated with t he degree of proliferation of MK progenitors induced by cytokines. In contrast, ploidy development closer to that observed in marrow MKs was observed in MKs derived from the low proliferative CD34(+) CD41(+) pr ogenitors and was associated with a twofold to threefold increment in platelet production per MK, As shown using this CD34(+) CD41(+) cell p opulation, PEG-rHuMGDF was required throughout the culture period to p otently promote platelet production, but was not involved directly in the process of platelet shedding. IL-3, SCF, and IL-6 alone had a very weak effect on proplatelet formation and platelet shedding. Surprisin gly, when used in combination, these cytokines elicited a degree of pl atelet production which was decreased only 2.4-fold in comparison with PEG-rHuMGDF. This suggests that proplatelet formation may be inhibite d by non-MK cells which contaminate the cultures when the entire CD34( +) cell population is used. Cultured platelets derived from PEG-rHuMGD F- or cytokine combination-stimulated cultures had similar ultrastruct ural features and a nearly similar response to activation by thrombin. The data show that this culture system may be useful to study the eff ects of cytokines and the role of polyploidization on platelet product ion and function. (C) 1998 by The American Society of Hematology.