Diversity of human granulopoietic precursor cells: separation of cells that form colonies in diffusion chambers (CFU-d) from populations of colony-forming cells in vitro (CFU-c) by velocity sedimentation.

Normal human bone marrow contains cells that form granulocytic colonies in fibrin clot diffusion chambers implanted intraperitoneally in sublethally irradiated mice (CFU-d) A series of experiments was performed to determine the relationship between CFU-d and the cells that form colonies in agar culture in vitro (CFU-c). Low-density bone marrow cells ( < 1 070 g/cm3) were separated by velocity sedimentation and colony-forming cells in each fraction assessed by culture in diffusion chambers and in soft agar. CFU-d sedimented more slowly than the vast majority of CFU-c, with a peak sedimentation velocity of 5.0 ± 0.4 mm/hr. Two different CFU-c populations could be distinguished. One was a rapidly sedimenting cell population (7.3 ± 09 mm/hr.) that formed colonies after 1 wk incubation in vitro. On day 14, however, colonies were derived from more slowly sedimenting cells that had a peak sedimentation velocity of 6.0 ± 0.5 mm/hr. Clusters (3-50 cells), scored on day 7 in agar culture, were a heterogeneous population, possibly derived from both day 7 and day 14 CFU-c. Mixing experiments did not show evidence of cell interaction that could explain the observed velocity differences. The velocity sedimentation profiles of colony-forming cells in DNA synthesis (S phase) and non-S phase were determined by assessment of the proportion of separated cells sensitive to pulse treatment with high specific activity 3H-thymidine (3H-TdR) prior to culture. The resuits excluded the possibility that the assays simply detected identical cells in various stages of the mitotic cycle. Rather, three different populations of precursor cells were distinguishable by the culture techniques employed. In a series of five unseparated normal bone marrow sampies, neutrophilic CFU-d had a small proportion in S phase (7% ± 10%) as measured by 3H-TdR suicide experiments. The average proportion of CFU-c in S phase was 21% ± 7% and 48% ± 4% for day 14 and day 7 CFU-c, respectively.