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A study of the erythropoietin requirements of erythroid progenitors in polycythemia vera Cashman, Johanne Dianne


Erythroid progenitor cells of the abnormal clone in polycythemia vera (PV) are capable of colony formation in vitro without the addition of erythropoietin, the regulatory hormone required for normal in vivo and-in vitro erythropoiesis. This property of "erythropoietin-independent" colony formation has been considered a marker for the abnormal clone in PV, although recent studies indicate that not all erythropoietic members of the clone may be capable of exhibiting this abnormal phenotype. The present studies were undertaken to investigate the level of maturation at which establishment of an erythropoietin-independent phenotype might be determined. A series of experiments was performed on the replated progeny of single primitive hemopoietic cells already committed to erythropoiesis (primitive BFU-E). First, conditions were established to maximize the number of erythroid colonies obtainable in secondary assays of replated primary colonies of primitive BFU-E origin. Time course studies and experiments with irradiated peripheral blood "feeder" cells treated in different ways established that results were best when primary colonies were allowed to grow for 9 days prior to replating and when 9 day old feeders stored at 4°C were included in the secondary assay medium. Second, a technique was developed for dividing such colonies between 2 secondary assay cultures. Experiments with normal primary colonies transferred to 2 secondary assays, both containing erythropoietin, showed that the variation between true replicates was random, indicating that the procedure used divided each primary colony equally. Third, it was shown that secondary assays to which no erythropoietin was added failed to support erythroid colony formation by progenitors present in normal 9 day old primary colonies. Finally, the distribution of erythropoietin-dependent and erythropoietin-independent phenotypes in individual colonies derived from primitive BFU-E from 5 patients with. PV was assessed by replating experiments. Most of the replated colonies from PV cultures that yielded erythroid colonies in secondary assays containing 3 units of erythropoietin per ml also produced some erythroid colonies in the paired replicate that contained < 0.01 units of erythropoietin per ml. However, fewer colonies were consistently obtained in the low erythropoietin cultures. These results indicate that in PV, most of the primitive erythroid bursts that generate phenotypically abnormal progeny capable of erythroid colony formation under conditions which are non-permissive for normal cells also produce significant numbers of progeny that are phenotypically normal in this respect. It is concluded that the capacity for erythropoietin-independent growth and maturation exhibited in vitro by terminally differentiating members of the abnormal clone in PV is not commonly fixed at or prior to the primitive BFU-E stage of erythropoietic cell development.

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