The Effect of Erythropoietin on Colonial Growth of Erythroid Precursor Cells In Vitro

Abstract

A method is described for the colonial growth, in semi-solid medium, of erythropoietin-responsive erythroid cell precursors. The erythroid cell precursors were isolated by immune hemolysis from fetal mouse liver. Both the number of precursor cells triggered to proliferate and differentiate, and the size of the erythropoietic colonies formed, are directly dependent upon the concentration of erythropoietin included in the culture.Erythropoiesis comprises an orderly progression of replication and differentiation starting from a pluripotential hemopoietic stem cell and concluding with the release of reticulocytes and erythrocytes into the circulation. Several in vitro culture methods have been described which transiently support erythropoiesis, either in suspension in liquid medium (1,2) (4,5). In order to further characterize the effects of erythropoietin on the proliferation and differentiation of individual precursor cells it is necessary to employ techniques capable of examining the progeny of individual precursors. In this study, a technique for the colonial growth of precursor cells in semi-solid medium is described. This technique permits analysis of the responsiveness of single precursor cells to the hormone. MATERIALS AND METHODSErythroid precursor cells from 12-to 13-day C57B1/6J fetalmouse livers were prepared by selective immune hemolysis of more mature erythroblasts and erythrocytes with rabbit antimouse erythrocyte antiserum in the presence of complement (4). These populations are contaminated with about 5% recognizable granulocyte precursors, macrophages, and hepatic epithelial cells.The components of the culture medium (6) are provided in immediately prior to use at a final concentration of 15%.The final viscous medium was transferred and distributed with a plastic syringe equipped with a 14 gauge 10-cm needle.Erythroid precursor cells were suspended at concentrations between 5 X 104 and 8 X 105 cells per-ml, in the final culture medium containing, in addition, 25,4I of a solution of erythropoietint at various concentrations'as indicated in the text. The suspension was mixed vigorously with the aid of a Vortex mixer, distributed in 1-ml aliquots into 35-mm plastic petri dishes (Falcon), and incubated at 370 in a humidified incubator gassed with 5% CO2 in air.The growth of erythroid colonies was scored by the examination of petri dish cultures, after selected periods of growth, with an inverted microscope equipped with a calibrated reticle. The compact form, and the small size of the individual cells in erythroid colonies serve to distinguish them from nonerythroid colonies in the culture (predominantly composed of macrophages and granulocyte precursors). These features were confirmed, initially, by the examination of colonies retrieved onto microscope slides and stained with benzidine-Wright-Giemsa (7) In order to further confirm the features used for identification of erythroid colonies, entire culture -plates were fixed in situ by flooding with 2 ml of 1% glutaraldehyde in 0.1 M phosphate ...

Stem cell expression of the AML1/ETO fusion protein induces a myeloproliferative disorder in mice

“…Myeloid progenitors (CFU-G, CFU-GM, CFU-M) were enumerated after 8 days of culture. The plates were then stained with benzidine, and burstforming units-erythroid (BFU-E) were counted, as described (21). Reticulocyte analysis was performed as described, with minor modifications (22).…”

Differential regulation of SOCS genes in normal and transformed erythroid cells

“…Cells were stimulated with recombinant human Epo (0.01-5 U/ml; Eprex, Jannsen-Cilag, Australia). Hemoglobin synthesis determined by benzidine staining (Cooper et al, 1974). [ 3 H]thymidine uptake assays were performed essentially as we have described previously (Busfield and Klinken, 1992).…”

Effective reversal of a transformed phenotype by retrovirus-mediated transfer of a ribozyme directed against mutant N-ras

“…32 A 5 l cell suspension was resuspended in 55 l IMDM, 10 l benzidine dihydrochloride (0.2%; Sigma, St Louis, MO, USA) and 1 l H 2 O 2 (30%). The percentage of bluestained cells was assessed with an hematocytometer after 5 min incubation at room temperature.…”