Separation of normal hemopoietic stem cells from clonogenic leukemic cells in a rat model for human acute myelocytic leukemia. II. Velocity sedimentation in combination with density gradient separation

In efforts to obtain optimal bone marrow grafts for autologous marrow transplantation in acute leukemia, the efficacy of velocity sedimentation in combination with density gradient centrifugation was investigated in a rat model for human acute myelocytic leukemia (BNML). As reported, normal hemopoietic stem cells (HSC; CFU(s)) and clonogenic leukemic cells (LCFU(s) have distinct sedimentation rate profiles (peaks at 3.5 and 7.0 mm/h, respectively). In the density gradient, CFU(s) settle in the 1.065-1.068 g/cm3 fractions, while the majority of leukemic cells have a density greater than 1.071 g/cm3. Below a density of 1.071 g/cm3, 34-47% of the HSC and a maximum of 10-20% of the leukemic cells are recovered. The two-step separation procedure was applied to suspensions containing 1 leukemic cell/200 normal marrow cells. If the density gradient separation preceded velocity sedimentation, the final yield of HSC was higher as compared with 'velocity' followed by density, i.e., 13-27% versus 2-11%. The repopulating capacity of the fractions, as a measure for the number of HSC after separation, was tested by injecting the stem cell fractions into lethally irradiated recipients. Of the rats 27-36% died from aplasia. Calculations of the minimum number of HSC required before separation to yield a sufficient number for transplantation are presented. Leukemic cells could be eliminated completely in 5 of 8 experiments, as judged by the leukemogenic potential of the postseparation cell suspension (elimination factor > 1500). Only a few leukemic cells were left in the remaining experiments: 1-50/3 x 105 marrow cells. It was concluded that the limit for effective separation is at a concentration of 0.5% leukemic cells, i.e., 1 leukemic cell/1.4 HSC. As the leukemic cell contamination is likely to be less during the phase of complete remission, these results are encouraging in terms of the possible preparation of leukemia free autologous marrow grafts.