THE PRODUCTION OF LARGE SIGNALING COMPETENT MYELOID CELLS FROM CIRCULATING CD34(+) CELLS IN NEONATAL BLOOD

A method is described for the production of large myeloid cells, expre ssing functional formylated peptide receptors. CD34(+) cells were isol ated from neonatal cord blood by a two stage cell sorting method. Incl usion of SCF, together with IL-3, GM-CSF or both cytokines stimulated growth of these cells over 14 day period. The resultant cells, which r anged from 30 mu m to 100 mu m in size, were maintained in culture for up to 5 weeks, during which time the cell population increasingly dis played myeloid characteristics, including expression of formylated pep tide receptors, phagocytosis and oxidase activation. These large cells had a functioning Ca2+ signalling system in response to the chemotact ic peptide, f-Met-Leu-Phe. The large size of the cells enabled the ris e in cytosolic free Ca2+ which resulted from either transmembrane infl ux of extracellular Ca2+ and release of Ca2+ from intracellular stores to be visualised. These large myeloid cells thus provide a model syst em for investigating the spatial characteristics of Ca2+ signalling by formylated peptide receptors on human myeloid cells.