SELECTIVE EXPANSION OF NORMAL HEMATOPOIETIC PROGENITORS FROM CHRONIC MYELOGENOUS LEUKEMIA MARROW

CD34(+) and CD34(+) DR-cells from the bone marrow (Bh?) of chronic-pha se chronic myelogenous leukaemia (CML) patients at diagnosis were test ed for their colony-forming ability in response to early and intermedi ate-late colony stimulating factors (CSFs), Molecular analysis reveale d 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 signifi cantly lower than those of comparable DR- cells from normal donors. Ho wever. clonogenic assays using recombinant human CSFs demonstrated a r emarkable 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, opti mal stimulation of CD34(+)DR(-) cells from normal donors required co-i ncubation with three or more CSFs. Stroma-noncontact long-term culture s were then established in the presence of exogenous CSFs and human ir radiated allogeneic stromal layers or the murine stromal cell line M2- 1OB4, engineered to produce G-CSF and IL-3, In these cultures the comb ination 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 primit ive 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 valu es. 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 ha ve a different pattern of response to CSFs than normal cells. In addit ion, we established culture conditions which allow selective expansion of benign haemopoietic cells coexisting with leukaemic progenitors.