Human megakaryocytic progenitors (CFU‐M) assayed in methylcellulose: Physical characteristics and requirements for growth

The basic culture requirements and several physical characteristics were defined for megakaryocytic colony‐forming cells (CFU‐M) from normal human marrow growing in methylcellulose. Ficoll‐hypaque separated mononuclear cells from human, marrow gave rise to megakaryocytic colonies in the presence of normal human plasma and phytohemagglutinin‐stimulated leukocyte‐conditioned medium (PHA‐LCM). Their identity as megakaryocytic colonies was confirmed by immunofluorescence staining with a monoclonal antibody to human factor VIII antigen and by electron microscopy of individually harvested colonies. Demonstration of the single‐cell origin of the colonies was provided by analysis of the glucose‐6‐phosphate dehydrogenase (G‐6‐PD) enzyme type of individually harvested colonies grown from a G‐6‐PD heterozygote. The colonies grew best in heparinized or citrated plasma as opposed to serum. Detailed studies suggested that platelet‐release products were responsible for this difference. Tritiated thymidine suicide studies showed that the percentage of CFU‐M in DNA synthesis was 23 ± 8% (n = 10). The modal velocity sedimentation rate of CFU‐M was 4.9 ± 0.6 mm/hr (n = 4) while that of concurrently studied granulocyte/macrophage colony‐forming cells (CFU‐GM) was 5.7 ± 0.5 mm/hr. Examination of the PHA‐LCM dose‐response characteristics suggested the presence in the conditioned medium of an inhibitor to megakaryocyte colony growth which was partially removed by chromatography of the medium on Sephadex G‐100. The resulting conditioned medium increased the cloning efficiency for CFU‐M compared with that with crude PHA‐LCM (15.3 ± 7.0 and 8.2 ± 5.3/105 marrow cells, respectively).