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Quantitation of human megakaryocyte progenitor cells in semi-solid cultures Fanning, Stephen Francis


In vitro assays for the study of human megakaryocyte (Mk) progenitor cells have been compromised by the exquisite sensitivity of these cells to transforming growth factor-ϐ (TGF-ϐ) (which is found in : inhibitory concentrations in serum and plasma) and a need to use immunocytochemical methodology to specifically identify clonal populations of Mk lineage cells. The present studies were undertaken to identify conditions that would overcome both of these problems and allow human Mk progenitors to be detected at optimal efficiency. The final procedure developed involved plating normal adult human bone marrow cells in chamber-slides in cell culture grade agarose dissolved in Iscove's medium supplemented with 1% albumin, 200 µg/ml iron-saturated transferrin, 10 µg/ml insulin, 40 mmol/L l ow density lipoproteins, 5 x 10⁻⁵M 2-mercaptoethanol and a growth factor cocktail consisting of IL-3, IL-6, GM-CSF and Steel factor. After 2 to 3 weeks at 37°C , cultures were fixed in methanol: acetone and colonies containing cells expressing glycoprotein IIb/IIIa identified by immunoperoxidase staining. Colonies containing increasing numbers of positively stained cells appeared after increasing periods of time consistent with the presence in normal human bone marrow of a hierarchy of Mk progenitor cells of differing proliferative potential. These conditions were found to be superior to serum- or plasmacontaining medium in their ability to support the proliferation of human Mk progenitor cells. Dose response studies were undertaken to confirm that the concentrations of albumin and 2-mercaptoethanol were optimal. For 2 of 3 bone marrows, the number of Mk colonies seen. with up to 80 mmol/L low density lipoproteins was the same as when these were not added to the medium. The growth factor combination of insulin, IL-3, IL-6, G M - C S F and Steel factor was superior to other combinations tested (including some containing G-CSF and/or IL-11) and provided maximal stimulation of the larger (>50 cells/colony) Mk colonies. Under these conditions, the Mk colony yield after 18 to 20 days was linearly related to the number of cells plated over a wide range of cell concentrations. These findings validate the use of this assay for future studies of the factors regulating both the development and subsequent differentiation of human Mk progenitor cells.

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