Individual BFU-E in polycythemia vera produce both erythropoietin dependent and independent progeny

Erythropoietic progenitors from peripheral blood of normal individuals or patients with polycythemia vera (PV) were cultured in methylcellulose medium containing 2.5 U/ml of erythropoietin (Ep). After 7-9 days, colonies considered to be early stage large bursts were individually removed, resuspended in a small volume of fresh methylcellulose medium, and then divided between 2 dishes. To one of these secondary cultures, sufficient Ep was added to bring the concentration of Ep up to approximately 3 U/ml. To the other was added an equal volume of medium but no Ep. The final concentration of Ep in these cultures was determined to be less than 0.01 U/ml. Nine days later, both types of secondary cultures were scored for the presence of colonies containing 8 or more hemoglobinized erythroblasts. Of 90 primary colonies from 3 normal individuals assessed in this way, 59 gave secondary erythroid colonies in the high Ep cultures, while none gave secondary erythroid colonies in the low Ep cultures. Additional control experiments in which primary colonies from normal individuals were divided into duplicate high Ep cultures showed that on average, the procedure used divided primary colonies equally. Of 109 primary colonies from 5 PV patients that yielded secondary erythroid colonies in the high Ep cultures, 21 yielded no secondary erythroid colonies in the low Ep cultures. The other 88 yielded erythroid colonies in both, but the secondary colonies in the low Ep cultures were consistently smaller in size and significantly fewer in number. Similar results were obtained when primary colonies were generated in cultures to which no Ep was added. These findings indicate that primitive BFU-E in patients with PV can be subdivided into 2 populations: a minor population restricted to the production of erythroid colony-forming cells (Ep-dependent progenitors) that require Ep for their detection, and a major population that is not restricted in this way. In addition, these experiments show that most of the primitive BFU-E that generate Ep-independent progenitors also produce significant numbers of cells that are Ep-dependent.