Some Characteristics of in Vitro Erythroid Colony and Burst-Forming Units
Regulatory mechanisms of erythropoiesis have been studied mainly in vivo. Erythropoietin (EP), thought to be the primary regulator, is defined by its erythropoiesis-stimulating properties in experimental animals. The target cells of EP are operationally t
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Regulatory mechanisms of erythropoiesis have been studied mainly in vivo. Erythropoietin (EP), thought to be the primary regulator, is defined by its erythropoiesis-stimulating properties in experimental animals. The target cells of EP are operationally termed erythropoietin-responsive cells (ERC) (6). They are considered to be primitive hemopoietic cells, not morphologically recognizable as erythroid, and induced by EP to form hemoglobin (16). ERC, in contrast to the maturing erythroid series, are present in erythropoiesissuppressed states such as transfusion- or hypoxia-induced polycythemia (11). The ERC seem to be more differentiated than the pluripotent hemopoietic cell population (CFU-s) detected by the in vivo spleen colony assay (3, 12). This population of target cells of EP is apparently a class of progenitor cells with a role in hemopoietic differentiation quite similar to that of the in vitro myeloid colony-forming cells (CFU-c). It has recently become possible to clone erythroid progenitor cells in vitro. Cui ture of mouse bone marrow cells in semisolid cultures containing EP detects two distinct populations of erythroid progenitor cells. One, termed CFU-e (erythroid colonyforming unit), forms a cluster of erythroid cells after 2-3 days of incubation (1, 5, 8, 19). The other forms a very large colony at 9-10 days of incubation (1, 10), which is termed a "burst," because of its dispersed appearance and explosive growth; the cells of origin are designated as BFU (burstforming units). It is the purpose of this chapter to describe some characteristics of BFU and CFU-e, and to discuss the applicability of cloning assays for erythroid progenitor cells as tools in studying the regulation of erythroid differentiation.
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METHODS AND MATERIALS
MICE Female BCBA F 1 mice, 8-10 weeks of age, were used for the experiments, unless otherwise stated. SPLEEN COLONY ASSAY CFU-s were estimated as described by Till and McCulloch (20). Briefly, mice were radiated with a supralethal dose of 1025 rad (y), and in-
103 S. J. Baum et al. (eds.), Experimental Hematology Today © Springer Science+Business Media New York 1977
Some Characteristics of in Vitro Erythroid Colony and Burst-Forming Units G. Wagemaker, V. E. Ober-Kieftenburg, A Brouwer, and M. F. Peters-Slough
Physiology of Committed Stem Cells (CFU-E and CFU-M)
jected with 2.5 and 5 x to• bone marrow cells. Macroscopic spleen colonies were counted tO days later after fixation with Telleyesnicki's solution. IN VITRO CULTURE OF HEMOPOIETIC PROGENITOR CELLS
Bone marrow cells were flushed from femurs with a-medium (8), and counted by hemocytometer using Turk's solution. The cells were cultured in a-medium, supplemented with tO% horse serum (Flow), tO% fetal calf serum (Flow), and tO% trypticase soy broth (BBL), using methylcellulose (4000 cps, Dow Chemical Int., U.S.A.) as a semisolidifying agent. For erythroid cultures, 10-• M 2-mercaptoethanol was added. Triplicate cultures, containing t ml in 3.2 em diameter tissue culture Petri dishes (Falcon Plastics), were incub
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