Contact-mediated inhibition of human haematopoietic progenitor cell proliferation may be conferred by stem cell antigen, CD34.

Abstract

The function of CD34, a transmembrane sialomucin expressed by human haematopoietic progenitor cells, is poorly understood. Its structure suggests it may act as a cell adhesion and signalling molecule. KGIa cells and primary CD34-positive marrow cells were tested for their ability to aggregate in the presence of the anti-CD34 antibody QBEND10; CFU-GM colonies were grown using standard methods and tested for their content of colony-forming cells by replating; 'haematons' were isolated from marrow by filtration; the phosphorylation of CD34 was investigated by immunoprecipitation and Western blotting CD34-positive cells in human bone marrow, like KG1a cells, aggregate when incubated with QBEND10. Staining aggregates with anti-CD34-FITC revealed that aggregation involved co-localisation of CD34 at intercellular binding sites. We examined myeloid colonies (CFU-GM) grown from normal human bone marrow cells, and multicellular aggregates ('haematons') separated from freshly aspirated marrow by filtration, and found CD34-positive cells bound together with co-localisation of the CD34 at the binding sites. This finding shows that CD34-positive cell-cell adhesion occurs physiologically in vitro and in vivo. QBEND10-induced aggregation of KG1a and CD34-positive cells was enhanced by staurosporine (a protein kinase C inhibitor) and inhibited by genistein (a protein tyrosine kinase inhibitor). Moreover, aggregated cells had increased phosphorylation of tyrosine on CD34 and translocation of protein kinase C (PKC) to the cytoplasm, compared with non-aggregated cells. We used the ability of primary colonies to produce secondary colonies on replating as a functional parameter and found that the replating ability of the colonies was increased by treatment with genistein (P=0.003). In addition, the ability of individual samples of primary CD34-positive cells to undergo QBEND10-induced aggregation and the ability of CD34-positive cell-derived colonies to produce secondary clones on replating were inversely related (r=0.86). Our results suggest that homotypic aggregation of haematopoietic progenitor cells may be an important mechanism for preventing inappropriate proliferation in vivo. Thus, regulation of expression of the CD34 molecule may play an important role in maintaining the normal level of haematopoietic activity by contact-mediated inhibition of progenitor cell proliferation.