Purification and Culture of Erythroid Progenitor Cells

Abstract

Purified cells can be utilized for studies involving growth factor regulated development of erythroid progenitor cells. For example, Fig. 4 demonstrates the expression pattern of β-globin and glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA during differentiation of highly purified human erythroid progenitors. Studies have been performed to delineate the expression of Epo receptor mRNA and the roles of each of the growth factors that are required for proliferation and differentiation of these cells [32, 33]. In addition, highly purified ECFC are useful for studying genes that regulate apoptosis since the withdrawal of Epo from these cell cultures induces apoptosis within 6–10 hours, between days 7–9 of culture [3,34]. Also, these cells are useful for studying erythroid membrane proteins and their structures, including biophysical properties in a dynamic fashion during terminal differentiation [35]. ECFC could also be utilized to extract RNA for construction of cDNA libraries in order to isolate novel genes that may play a role in guiding the differentiation of erythroid progenitors. Finally, the methodologies reported here can be used to purify cells from hematopoietic progenitor cell disorders such as PV [ 18,24], myelodysplasia, and erythroleukemia for studying genes involving the pathogenesis of these diseases. The study of the development of normal human erythroid progenitor cells, as well as diseased human progenitor cells, depends on the purity, uniformity of maturation, and the number of these target cells. Pure cells are just as vital to good studies as pure chemicals, and studies of primary cells are extremely important to verify real mechanisms and to remove the possibility of artifacts due to transformed cell lines. It is now clear that the methods described here for the study of human blood BFU-E and CFU-E allow molecular studies to proceed that should reveal the normal control of erythropoietic development, as well as the pathogenesis of erythropoietic diseases.