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 CD 34+ 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.
Fogli
M
M
Institute of Haematology and Medical Oncology Seràgnoli, University of Bologna, Italy.
Amabile
M
M
Martinelli
G
G
Fortuna
A
A
Rondelli
D
D
Ratta
M
M
Curti
A
A
Tura
S
S
Lemoli
R M
RM
D018952:Antigens, CD34 / Q000378:metabolism; D001854:Bone Marrow Cells / Q000473:pathology*; D002454:Cell Differentiation / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D002555:Cerebrospinal Fluid / Q000502:physiology; D016044:Fusion Proteins, bcr-abl; D006412:Hematopoietic Stem Cells / Q000473:pathology*; D006801:Humans; D015466:Leukemia, Myeloid, Chronic-Phase / Q000378:metabolism / Q000473:pathology*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014410:Tumor Stem Cell Assay
D018952:Antigens, CD34; D012333:RNA, Messenger; D016044:Fusion Proteins, bcr-abl