A culture method has been developed in which erythroid colonies are produced in vitro from hemopoietic cells from the livers of 13-day fetuses of C3Hf/Bi mice. Heme synthesis by the cultures was correlated with the presence of these colonies, and the hemoglobin produced was shown to be electrophoretically normal. The individual colonies were identified as erythroid since they were erythropoietin-dependent, positively stained by the histochemical 'Lepehne' procedure for hemoglobin, and labeled by 59Fe radioautography. Evidence is presented that the development of these colonies is under separate control from that of granulocytic colonies found in the same cultures.

An improved plasma culture system is described for the production of erythrocytic colonies by mammalian adult hemopoietic cells in vitro under the influence of erythropoietin. The concentration of fetal calf serum in the medium used for dilution of the cells was critical for erythrocytic colony formation when low numbers of cells were plated. Optimal concentrations were found for plasma, fetal calf serum, bovine serum albumin, and L-asparagine in the culture medium, and the colony-forming efficiency was shown to depend on the concentration of erythropoietin. With erythropoietin at plateau concentration, the number of erythrocytic colonies produced was directly proportional to the number of bone marrow or spleen cells plated, over a wide range of cell concentrations. Colony numbers per culture conformed to a Poisson distribution. Thus, the improved plasma culture system may be used for the quantitative assay of CFU-E. The method is rapid (2 days), reliable, convenient, and inexpensive. Since the improved plasma culture system also supports granulocytic colony formation by bone marrow cells in the presence of conditioned medium (CSA), and the number of granulocytic colonies produced is proportional to the number of cells plated, the same hemopoietic cell suspensions can be simultaneously assayed for CFU-E and CFU-C under virtually identical conditions.

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Interleukin 3 (IL-3) is a regulatory glycoprotein required for the proliferation and differentiation of cells from many if not all hemopoietic lineages. With the emergence of the competence-progression model of cell proliferation, which predicts that growth factors function at specific stages of the cell cycle, we examined the possibility that IL-3 functions at a specific stage of the cell cycle. C-63 cells were developed as a cell line from normal murine bone marrow. They have a mast cell phenotype and require pokeweed-stimulated spleen cell-conditioned medium (CM), a rich source of IL-3, for their continued growth. Exponentially growing cells were transferred from growth medium, which contains CM, to medium lacking CM or IL-3. After 24 hours, cell viability had decreased 40-50%. The remaining viable cells did not incorporate 3H-thymidine, and displayed a single peak at G1 in a DNA histogram. Restimulation of these cells with CM or IL-3 resulted in a dramatic rise in 3H-thymidine uptake 20-24 hours after restimulation. DNA histograms of restimulated cultures indicated that the cells were progressing in a wave-like fashion throughout the remainder of the cell cycle. The length of time necessary for cells to be in contact with CM or IL-3 before they could progress into the remainder of the cell cycle was also examined. Cells incubated with CM or IL-3 for less than 16 hours could not progress into S phase, whereas cells incubated for 16 hours or longer could progress into S phase and through the remainder of the cell cycle. These data suggest that IL-3 exerts its function at a specific stage of the cell cycle.

Replication of multipotential stem cells in long-term murine bone marrow cell culture is known to depend on the development of an adherent stromal cell layer. In these conditions, restricted haematopoietic progenitor cells have also been generated for up to several months1-3. However, maturation is observed only in the granulocyte/macrophage and megakaryocyte lineages; erythropoiesis appears to be blocked at the earliest burst-forming unit (BFU-E) stage. Addition of exogenous erythropoietin (Epo) or anaemic mouse serum results in full erythropoietic maturation, but it is transient. We describe here a culture system in which production of erythropoietic progenitor cells can be maintained for over 6 months in the absence of an adherent stromal layer and in the absence of added Epo, but in the presence of pokeweed mitogen-stimulated spleen cell conditioned medium (PWSCM). The data indicate that restricted erythroid progenitor cells exist which are capable of extensive self-renewal.