Effects of BMP2 and BMP4 treatments on leukemic progenitor amplification. Selected CD34+ cells isolated from healthy donor (open bars) or CP-CML (closed bars) samples were incubated in serum-free medium (6 × 105/mL) for 7 days in the presence of BMP2 or BMP4 (50 ng/mL). *P < .05 indicate differences between healthy and CML samples. Cell viability (A) and proliferation (B) were evaluated using trypan blue staining and cell counting. Results are expressed as the percentage of viable cells or the proliferation ratio, which is the ratio of cell counts to the number of input cells. The mean ± SEM of 10 experiments is presented. (C) Phenotypic analysis of CD34, the hematopoietic progenitor marker, was performed using flow cytometry on a FACSCalibur cell analyzer (Becton Dickinson) and gated for viable cells. Results are presented as the mean ± SEM of 5 experiments for healthy donors and 8 experiments for CML patients. (D) SC content of treated cells was analyzed by the LTC-IC coculture assay, and results are presented as the mean value ± SEM for 12 or 9 experiments for healthy donors and CML patients, respectively. Results are presented as the total LTC-IC–derived colonies after 5 weeks of coculture per 1 × 104 seeded cells. (E) The progenitor content of treated cells was analyzed by the clonogenic CFC assay. Results are expressed as the ratio of treated to untreated cells and represent the mean value ± SEM of 12 or 19 experiments for healthy donors and CML patients, respectively. (F) The number of leukemic colonies in either CFC- or LTC-IC–derived colonies was assessed by picking individual colonies to quantify expression of ABL, BCR-ABL, and the reference genes BGUS and TBP by qPCR. Leukemic positive colonies were determined by simultaneous detection of all genes, whereas normal negative colonies were stated as ABL-positive but BCR-ABL–negative. In all cases, internal gene controls were positive; otherwise, those samples would have been removed. Results are expressed as the percentage of positive BCR-ABL colonies of the total assayed colonies of 3 experiments for healthy donors and CML patients.
