• pH;
  • ex vivo expansion ;
  • erythroid differentiation

Physiological parameters such as pH and oxygen tension probably play significant roles in the regulation of haemopoiesis in the bone marrow microenvironment, but these roles have yet to be characterized in detail. We have found that changes in culture pH (0.2 units) can cause significant changes in the culture composition of mature cells and colony-forming cells (CFCs), especially in the presence of erythropoietin (Epo). Peripheral blood (PB) CD34+ cells cultured at different pH values (7.15–7.6) were characterized using total cell counts, colony assays, morphological analysis, haemoglobin staining, flow cytometry, immunocytochemical staining, and Western blots. Cultures performed at high (7.6) pH contained greater numbers of haemoglobin-positive and band-3-positive cells, and acquired these erythroid differentiation markers sooner than standard (7.35) and low (7.1) pH cultures. Flow cytometry using CD71 and CD45RA antigens also indicated that erythroid differentiation proceeds faster at high pH and is blocked at an intermediate stage by low pH. Morphological data confirmed that high pH cultures had been shifted towards late-stage erythroid compartments as compared to low and standard pH cultures. These findings have important implications both in elucidating the regulatory role of pH in the bone marrow microenvironment and for the design of in vitro systems to study the development of erythroid cells.