Blast cell colony assay for umbilical cord blood and adult bone marrow progenitors

We previously described candidate human blast cell colonies in culture of umbilical cord blood cells. However, their replating efficiencies were low, and we were unable to grow colonies from adult marrow cells. We report here a consistent method of growth and identification of human blast cell colonies that are supported by low serum culture and by delayed addition of medium conditioned by a T lymphoblast cell line, C5MJ. Nonadherent mononuclear cord blood and bone marrow cells were prepared by use of Ficoll-Paque and overnight adherence to plastic. Bone marrow cells were further enriched for progenitors by panning with monoclonal anti-My-10 antibody. Cells were plated in methylcellulose culture containing 2% fetal calf serum and supplemented with bovine serum albumin, lecithin, cholesterol, and transferrin. On day 14 of culture, concentrated C5MJ-conditioned medium was carefully added to each dish. Blast cell colonies consisting of 18 to 100 cells were detected on days 21 to 28. Forty percent to 75% of the blast cell colonies in individual samples yielded secondary colonies upon replating (positive colonies). The replating efficiency of the positive colonies ranged from 3% to 100%. The largest secondary colony contained 7,800 cells. In addition to single-lineage colonies, multilineage colonies revealing two to five lineage combinations were seen. These results suggest that human primitive progenitors are dormant in cell cycle and that they survive in the absence of colony-stimulating factors. Human blast cell colonies may provide a unique population of progenitors for studies of the early process of human hemopoiesis.