Purification of Erythropoietin-Responsive Cells by Immune Hemolysis

Abstract

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...

Potentiation of erythropoiesis in vitro by dexamethasone.

“…Erythropoietin is thought to act via a specific receptor mechanism on certain red cell progenitors (2,27). The present studies suggest that these cells may also have a glucocorticoid receptor mechanism.…”

“…Cortisone has been found inhibitory to granulopoiesis both in vivo and in vitro (26). Mouse fetal liver is a rich source of erythropoietinresponsive cells (27) and has been used in assays for erythropoietin (28). We used a cloning assay employing fetal liver cells in order to assess the effect of dexamethasone on mammalian erythropoiesis in vitro.…”

Globin Messenger RNA Activity in Erythroid Precursor Cells and the Effect of Erythropoietin

“…[4,5- (2). These precursor cells were cultured, with or without erythropoietin, according to methods detailed elsewhere (2). In order to determine the rate of synthesis of total proteins and of globin, cells were suspended in leucine-free Waymouth's medium with [14C]leucine, supplemented with dialyzed 10% fetal-calf serum, four ribonucleosides (UCG, and A) at 1 ,ug/ml, four deoxyribonucleosides (dT, dC, dG, and dA) at 10 g/ml, and 3.6 ;&g/ml 750 X g for 3 min at 4°.…”

“…[4,5- (2). These precursor cells were cultured, with or without erythropoietin, according to methods detailed elsewhere (2).…”

“…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.…”