Colony formation in agar by adult bone marrow multipotential hemopoietic cells

Erythroid colony formation in agar cultures of CBA bone marrow cells was stimulated by the addition of pokeweed mitogen‐stimulated spleen conditioned medium (SCM). Optimal colony numbers were obtained when cultures contained 20% fetal calf serum and concentrated spleen conditioned medium. By 7 days of incubation, large burst or unicentric erythroid colonies occurred at a maximum frequency of 40–50 per 10 5 bone marrow cells. In CBA mice the cells forming erythroid colonies were also present in the spleen, peripheral blood, and within individual spleen colonies. A marked strain variation was noted with CBA mice having the highest levels of erythroid colony‐forming cells. In CBA mice erythroid colony‐forming cells were mainly non‐cycling (12.5% reduction in colony numbers after incubation with hydroxyurea or 3H‐thymidine). Erythroid colony‐forming cells sedimented with a peak of 4.5 mm/hr, compared with CFU‐S, which sedimented at 4.25 mm/hr. The addition of erythropoietin (up to 4 units) to cultures containing SCM did not alter the number or degree of hemoglobinisation of erythroid colonies. Analysis of the total number of erythroid colony‐forming cells and CFU‐S in 90 individual spleen colonies gave a correlation coefficient of r = 0.93 for these two cell types. In addition to benzidine‐positive erythroid cells, up to 40% of the colonies contained, in addition, varying proportions of neutrophils, macrophages, eosinophils, and megakaryocytes. Taken together with the close correlation between the numbers of CFU‐S in different adult hemopoietic tissues, including individual spleen colonies, the data indicate that the erythroid colony‐forming cells expressing multiple hemopoietic differentiation are members of the hemopoietic multipotential stem cell compartment.