System for the ex vivo development of adult bone marrow-derived erythroblasts. (A) Outlined are steps used in the serum-free expansion and MACS-based isolation of marrow-derived erythroid progenitor cells. (B) Flow cytometric, cytospins, and colony-forming features of isolated erythroid progenitor cells. (i) Upon expansion for 4.5 days, cultures routinely were composed of approximately 50% to 60% Kit^posCD71^high and approximately 40% to 50% Kit^negCD71^high erythroblasts. (ii-iii) Wright-Giemsa staining of isolated Kit^negCD71^high and Kit^posCD71^high erythroblasts. (iv) Colony morphologies of Kit^posCD71^high cells cultured for 2.5 days in 0.9% methylcellulose with Epo at 5 U/mL. (C) Differentiation capacities of expanded erythroblasts. Kit^posCD71^high cells were isolated (by MACS) from expansion cultures and transferred to BSA, insulin, and transferrin (BIT)-containing medium. At 24 hours of culture, few cells continued to express Kit (CD117) while more than 50% routinely differentiated to Ter119^pos erythroblasts (lower right panel, cytospin preparation).