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 ...
Cells transformed by Friend virus in liquid suspension culture respond to low concentrations of dimethylsulfoxide by initiating hemoglobin synthesis. The kinetics of appearance of such differentiated erythroid cells is consistent with either the induction of differentiation in a uniformly susceptible population of transformed cells or selection for the growth of a distinct erythropoietic subpopulation. The dimethylsulfoxide response of individual colonies of cells transformed by Friend virus grown in semisolid medium was studied in order to distinguish between these alternatives. The data do not support a selective effect of dimethylsulfoxide on the growth of a unique erythropoietic subpopulation; they indicate, rather, that the 745A strain of cells transformed by Friend virus consists of a relatively uniform population of dimethylsulfoxide-sensitive erythropoietic cells.
An increased incidence of lymphoid neoplasias is associated with the states of immune deficiency, both congenital and acquired. Twenty-one cases of lymphoma in men at high risk for Acquired Immune Deficiency Syndrome (AIDS) were diagnosed in one community hospital in New York City within the last 2 years. The mean age of these patients was 39.6 years, 20 were homosexual, and 1 was an intravenous drug abuser. There were 3 Hodgkin's and 18 non-Hodgkin's lymphomas of various histologic types, but almost all of high-grade categories. The proportion of extranodal lymphomas, the involvement of the gastrointestinal tract, central nervous system, bone marrow, and myocardium were significantly higher than in the lymphomas of the general population. The phenotypes were B-cell and non-B-non-T-cell types without any T-cell lymphomas. All patients had reversed helper-suppressor T-cell ratios and all those tested had circulating HTLV-III antibodies. Seven patients have had previous lymph node biopsies performed, showing the lesions of AIDS-related lymphadenopathies that often were directly associated with lymphoma. A variety of severe opportunistic infections and Kaposi's sarcoma affected these patients. All lymphomas in this group were highly aggressive, involved multiple organs, and responded poorly to treatment, resulting in early deaths.
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