Purified unfractionated G-CSF/chemotherapy mobilized CD34+ peripheral blood progenitors and not bone marrow CD34+ progenitors undergo selective erythroid differentiation in liquid culture in the presence of erythropoietin and stem cell factor.

Abstract

A combination of erythropoietin (EPO) plus stem cell factor (SCF) drove purified unfractionated granulocyte colony stimulating factor (G-CSF)/chemotherapy mobilized peripheral blood CD34+ cells to selective erythroid differentiation in liquid culture with an average 28-fold increase in the total cell number after 21 d. From day 6 of culture cytologic and cytofluorimetric characterization revealed that cultured cells belonged to the erythroid lineage with a gradual wave of maturation along the erythroid pathway to terminal cells. A similar pattern of erythroid differentiation was observed when the same peripheral blood CD34+ cells were culture with EPO plus SCF in serum-free medium. This cytokine combination produced selective erythroid differentiation with the complete exhaustion of the clonogenic potential on day 21. In parallel experiments the same circulating CD34+ cells underwent granulocytic/ monocytic differentiation in liquid culture in response to granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and SCF, demonstrating that these CD34+ progenitors had intact pluripotent differentiating potential. Conversely, bone marrow CD34+ cells isolated from bone marrow allografts were unable to selectively differentiate along the erythroid pathway when they were exposed to EPO plus SCF combination. However, these cells maintained a greater number of colony forming cells on day 21 of culture compared to mobilized peripheral blood CD34+ cells. This model is a simple and reliable way to obtain selective erythroid differentiation of peripheral blood G-CSF/ chemotherapy mobilized CD34+ progenitor cells in liquid culture. The absence of cytokines such as GM-CSF and IL-3 in the culture medium permits studies on in vitro erythropoiesis without disturbance of prevalent myelopoiesis.