[Terminal differentiation of human peripheral blood CD34 positive cells to reticulocytes in vitro and effects of cytoskeletal modifiers on enucleation]

Abstract

We have developed a system of erythroid-lineage-specific expansion of purified human peripheral blood (PB) CD34 positive cells mobilized by a granulocyte-colony stimulating factor (G-CSF), as an in vitro model for the study of the process of proliferation and differentiation of erythroid progenitor cells. In this system, PB CD34 positive cells terminally differentiated into reticulocytes, which made it feasible to conduct a study on enucleation process of human erythroblasts. Erythroid differentiation/maturation was induced in the highly purified PB CD34 positive cells in the liquid suspension culture with interleukin-3 (IL-3), stem cell factor (SCF; a ligand for c-kit) and human erythropoietin (EP); 8 days of the culture generated progenitors equivalent to colony-forming units-erythroid (CFU-E) and 12 days of the culture generated a population mainly consisting of polychromatophilic normoblasts. Within additional 4 days of the suspension culture, these cells contained hemoglobin, differentiated to orthochromatic normoblasts, and became capable of enucleation in vitro, in a time-dependent manner. Removal of all serum from the culture medium, with or without cytokines, including IL-3, SCF and EP, did not affect enucleation processes of the cells on 12th day. On electron microscopy, the incipient reticulocytes contained all cellular organelles except the nucleus, and the extruding nucleus was surrounded by a plasma membrane. Colchicine and vinblastine blocked nuclear multiplication and cytochalasin D blocked cell division with formation of multinucleated cells. Only cytochalasin D completely inhibited enucleation, which was recovered by washing out the cytochalasin D in the 12th day cell cultures. Thus, human erythroblasts do not require cytokines, including EP in their enucleation process. In this process, the contraction of filamentous actin occurs, while microtubules apparently do not participate.