Selective expansion of normal haemopoietic progenitors from chronic myelogenous leukaemia marrow

CD34⁺ and CD34⁺ DR^? cells from the bone marrow (BM) of chronic-phase chronic myelogenous leukaemia (CML) patients at diagnosis were tested for their colony-forming ability in response to early and intermediate-late colony stimulating factors (CSFs). Molecular analysis revealed that 55.6 ± 9% SD of CD34⁺DR^? colonies, in which actin and ABL mRNA were detectable, expressed the product of the BCR-ABL gene. The percentage and the clonogenic efficiency of CML DR^? cells were significantly lower than those of comparable DR^? cells from normal donors. However, clonogenic assays using recombinant human CSFs demonstrated a remarkable proliferation of CML cells when stimulated by SCF, IL-11 and IL-3, used as single factors in the presence of erythropoietin (EPO) and was almost entirely due to erythroid progenitors. Conversely, optimal stimulation of CD34⁺DR^? cells from normal donors required co-incubation with three or more CSFs. Stroma-noncontact long-term cultures were then established in the presence of exogenous CSFs and human irradiated allogeneic stromal layers or the murine stromal cell line M2-10B4, engineered to produce G-CSF and IL-3. In these cultures the combination of SCF and IL-3 induced a 25.4 ± 5 SD, 40 ± 6 SD and 20.5 ± 6 SD fold increase of colony-forming unit cells (CFU-C), at weeks 2, 4 and 5, respectively. At the same time-points the number of primitive long-term culture initiating cells (LTC-IC) showed a 4 ± 2 SD, 3.3 ± 1.5 SD and 2.3 ± 1 SD fold increase compared to baseline values. BCR-ABL mRNA analysis of single colonies demonstrated that 27 ± 9% SD and 7 ± 3% SD CFU-C at weeks 4 and 5, respectively, expressed the fusion gene, whereas leukaemic LTC-IC disappeared from the culture by week 2. These results suggest that leukaemic CD34⁺DR^? cells have a different pattern of response to CSFs than normal cells. In addition, we established culture conditions which allow selective expansion of benign haemopoietic cells coexisting with leukaemic progenitors.