Cells with marrow and spleen repopulating ability and forming spleen colonies on day 16, 12, and 8 are sequentially ordered on the basis of increasing rhodamine 123 retention.

Mouse bone marrow cells (BMC) were subjected to countercurrent centrifugal elutriation and subsequently separated on the basis of light scatter and fluorescence intensity after being labeled with the supravital dye Rhodamine 123 (Rh-123). The sorted cells were then assayed for their in vivo spleen colony-forming ability (day -8, -12, and -16 CFU-S) and their ability to repopulate the bone marrow or spleen over a 13-day period with CFU-S-12, CFU-GM, or nucleated cells. Cells with marrow repopulating ability (MRA), as measured by the ability of the sorted cells to repopulate the marrow with secondary CFU-S-12 or CFU-GM, had low affinity for Rh-123. These cells showed minimal spleen colony-forming ability, and the ratio of MRA to CFU-S-12 in this preparation was 309. Cells with spleen repopulating ability (SRA), CFU-S-16, CFU-S-12, and CFU-S-8 retained increasing amounts of Rh-123, respectively, and CFU-S-8 were almost exclusively found among cells with high Rh-123 affinity. These cells also included about half of all day-12 CFU-S, and the ratio of MRA to day-12 CFU-S was 0. The results show that MRA cells, SRA cells, CFU-S-16, CFU-S-12, and CFU-S-8 can be sequentially ordered on the basis of increasing mitochondrial activity. The data also demonstrate for the first time, and without the application of negative selection by the use of cytostatic agents, that MRA cells are a separate class of primitive hemopoietic stem cells that fully meet the criteria of pre-CFU-S.