We have used flow cytometry to quantitate nuclear c-myc protein, at each phase of the cell cycle, during in-vitro differentiation of CD34-positive stem cells isolated from normal human bone marrow by the monoclonal antibody, MY10. Mean c-myc protein levels in CD34-positive cells, consisting of >70% blasts, are lower than a marrow fraction containing myeloid cells of intermediate maturation, but have an invariant proportional relationship, with regard to nuclear mass, over the cell cycle. The majority of these primitive cells are non-cycling, as revealed by DNA content. Under our assay conditions, nuclear c-myc protein distribution over the cell cycle did not change as these progenitors entered a proliferative phase in culture. In cultures containing factors supporting myeloid maturation, mean G0G1 p62c-myc levels initially decline, then rise above starting values as promyelocytes and myelocytes differentiate from CD34-positive cells, and as proliferation begins. With further myeloid maturation, and while cell numbers are increasing, c-myc protein continues to increase. C-myc protein kinetics differ in cultures in which macrophages, rather than myeloid cells, predominate. These data indicate that a complex relationship exists between c-myc gene expression and proliferation, maturation and lineage in haemopoietic cells, and lend support to the notion that early down regulation may be causally associated with the differentiation process.

A method is described for the measurement of DNA index and cell cycle distribution in purified erythroid and myeloid populations from human bone marrow. Erythroid cells were prepared after complement mediated lysis of non-erythroid marrow cells. Myeloid cells were obtained by fluorescence activated cell sorting by forward and wide angle light scatter. Mononuclear marrow cells were prepared with a density gradient. Nuclei prepared from the separated populations were stained with propidium iodide. Myeloid cells had a higher DNA index than erythroid cells, and the mononuclear preparation had an intermediate value. There were more erythroid than myeloid cells in the S and G2M phases of the cell cycle. These lineage differences are particularly relevant when considering data derived from unseparated bone marrow cells, and further experiments are needed to determine the origin of these anomalies.

The erythroblasts from four normal bone marrows were enriched by using an anti-myeloid monoclonal antibody (TG-1), and a polyclonal antibody against mononuclear cells to effect complement mediated lysis of unwanted cells. The erythroblasts were cultured for 2 h in medium containing [59Fe]transferrin and [3H]-leucine, then fractionated on Percoll gradients according to their density. Whole cell iron uptake by early erythroblasts was greater than in the dense late erythroblasts. In all marrows, iron uptake into ferritin was highest in fractions containing the earliest erythroblasts and decreased with increasing erythroblast maturity. In three of the four marrows this paralleled the pattern of ferritin synthesis. It seems likely that apoferritin is synthesized in response to the amount of iron taken into the cell and this iron is incorporated within the protein shell to form ferritin.

Erythroblasts were enriched from human bone marrow samples and fractionated on Percoll gradients according to maturity. Heart-type and spleen-type ferritin was measured in each fraction by an immunoradiometric assay. In normal marrow, heart-type ferritin content was higher in the early erythroblast fractions and fell with maturation. Spleen-type ferritin content showed no such consistent change. Megaloblastic erythroblasts had a significantly higher ferritin content.