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 ...

Immature erythroid cell precursors from 12-and 13-day fetal mouse livers were concentrated by differential antibody-mediated hemolysis. The effect of erythropoietin on these purified erythroid cell precursors was studied in vitro. Addition of erythropoietin stimulates erythroid cells to proliferate, and to differentiate to cells that actively synthesize hemoglobin. This erythropoietininduced erythropoiesis is sustained in culture for at least 48 hr.Elucidation of the cellular and molecular effects of erythropoietin has been aided by the development of several in vitro systems, including the short-term culture of bone marrow (1) and fetal hepatic erythroid cells (2, 3). However, interpretation of these studies is hindered by the presence of large numbers of differentiated hemoglobin-synthesizing cells in the culture. Although such cells constitute about 70% of the total cell population in the 13-day fetal mouse liver, previous studies (2) indicate that in this system the primary target cell responsive to erythropoietin is a large, nonhemoglobinized precursor, morphologically indistinguishable from the cells called proerythroblasts, that constitutes less than 10% of the population. The present paper describes a method that uses immunological hemolysis (4-6) to concentrate immature erythroid cell precursors from the total erythropoietic population of fetal mouse liver. This immature cell population responds to erythropoietin by proliferation of erythroid cell precursors, which differentiate to hemoglobin-synthesizing cells. MATERIALS AND METHODSThe present procedures for the preparation of antibody to mature mouse erythrocytes are modified from earlier studies of Borsook et al. (4, 5) with antiserum to rabbit erythrocytes and of Minio-Paluello et al. (6) with antiserum to mouse erythrocytes. Rabbits were immunized with erythrocytes obtained from adult C57BL/6J female mice bled by cardiac puncture under ether anesthesia. Heparinized mouse blood was pooled, washed three times with saline, packed, and diluted 1:1 with saline; penicillin was then added. 1-1.5 ml Of the erythrocyte suspension was injected intravenously into the rabbits, via the ear vein, three times a week for 4 weeks. 7 Days after the last injection, the rabbits were bled by heart puncture and the serum was sterilized by Millipore filtration (0.45,um pore size). The sera were tested for complement-dependent hemolysin titer (1: 4000 or greater in each of three antisera prepared, all of which proved satisfactory for differential cell lysis). Rabbit sera were not subjected to heat inactivation.Timed pregnancies were obtained in hormonally primed C57BL/6J female mice (7); fetal hepatic hemopoietic cells were obtained from 12-or 13-day embryos by mechanical disaggregation of the dissected livers (3). Cells were collected in ice-cold Waymouth MB752/1 medium (GIBCO Grand Island, N.Y.), supplemented with 10% fetal-calf serum (Microbiological Associates, Bethesda, Md.), 3% chickembryo extract (GIBCO), and 1% penicillin-streptomycin (5000 units of peni...

Cell populations enriched for erythroid precursor cells were fractionated from 13-day fetal-mouse livers by a method of immune hemolysis. These preparations of precursors, contaminated by less than 7% hemoglobinized erythroblasts, synthesize globin at a rate less than 6% that of the unfractionated liver erythroid cell population. RNA was isolated from these precursor cells and assayed for globin mRNA activity in a cell-free system from Krebs ascites tumor. The 6-16S RNA fraction from precursor cells has less than 5% of the globin mRNA activity of RNA isolated from unfractionated populations. Precursor cells incubated with erythropoietin show an increment in the rate of synthesis of globin only after 5-10 hr of incubation. After 10 hr of culture with this hormone, precursor cells show a 6-to 10-fold increase in globin mRNA activity. These results suggest that the precursor cells of hepatic erythropoiesis, responsive to erythropoietin, do not contain globin mRNA in a biologically active form. Erythropoietin-induced differentiation of these cells to erythroblasts is associated with an increase in globin mRNA.Fetal-mouse liver erythropoiesis provides a model for study of mammalian cell differentiation (1). The erythroid cell population obtained from 13-day fetal livers contains cells at all stages of differentiation, from the immature, erythropoietinresponsive precursor to the fully hemoglobinized erythrocyte. Erythroid precursor cells can be concentrated from the livers of fetal mice of 12-and 13-days gestation (2). Incubation of these precursor cells with erythropoietin stimulates both proliferation and differentiation to cells that actively synthesize hemoglobin. The questions posed in the present study are: (i) Does the precursor cell population contain biologically active mRNA for globin and synthesize globin, and (ii) Does erythropoietin-induced stimulation of erythroid cell differentiation involve an increase in the amount of biologically active globin mRNA? The evidence indicates that precursor cell populations (contaminated with 2-7% hemoglobinized erythroblasts) contain less than 5% of the globin mRNA activity recovered from an unfractionated erythroid cell population from fetal liver, and that they synthesize globin at a rate less than 5% that of the unfractionated cells.Erythropoietin-induced differentiation of precursor cells is characterized by a marked increase in globin mRNA activity and in the rate of globin synthesis. (pH 7.4) and centrifuged at 750 X g for 3 min at 4°. The supernatant was removed, the nuclear fraction was washed once with 0.5 ml of this buffer, and the wash was combined with the first supernatant solution. (b) Mechanical lysis: the cells were suspended in 2 ml of 30 mM Tris-HCl-5 mM Mg acetate-10 mM KCl (pH 7.5) and lysed in a Dounce homogenizer with five strokes of the lightly-fitted pestle (5).

No abstract