Purification and characterisation of myeloid blast cells from human fetal liver and studies of changes in inositol metabolism during their differentiation towards monocytes

• Main Author: Patton, W. Nigel
• Published: University of Edinburgh 1995
• Subjects: 611
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.660446

A homogeneous population of myeloid blast cells was purified by negative selection from human fetal liver by enzyme digestion of liver tissue, ficoll fractionation, indirect erythrocyte rosette sedimentation of unwanted cells, after coating these cells with monoclonal antibodies directed against erythroblasts and macrophages, and finally by cell elutriation. Characterisation of these cells confirmed their undifferentiated status and in culture these cells generated only neutrophils and macrophages. Treatment of these cells with 10nM phorbol myristate acetate (PMA) induced rapid terminal monocyte differentiation of 62% of this population and also completely inhibited the neutrophil differentiation of the remaining cells. The undifferentiated blast cells were maintained in culture in a serum-free medium containing 100 U/ml interleukin-3 and 1mg/l inositol which permitted the equilibrium labelling of cells with [<SUP>3</SUP>H] myo-inositol and the subsequent analysis of concentrations of inositol metabolites within these cells. High concentrations of various inositol metabolites, similar to those found in HL60 cells, were observed in normal myeloid blast cells and following PMA-induced monocyte differentiation of these cells significant changes occurred - namely a decrease in inositol tetrakisphosphate (InsP<SUB>4</SUB>) and inositol pentakisphosphate (InsP<SUB>5</SUB>) and an increase in glycerophospho-inositol (GPI). These changes in response to PMA, with the exception of the rise in GPI, are similar to those reported in HL60 cells undergoing monocyte differentiation and suggest that abundant inositol polyphosphates may play an important role in myeloid differentiation.