IK6 consistently expands primitive CP-CML cell numbers more than normal cells in vitro. (A) Total mature (nonadherent) cells present in 2-, 4-, and 6-week-old cultures initiated with CD34⁺ cells (5-10 × 10³ each, depending on the experiment) from 5 CP-CML patients and 2 normal BM samples according to the design shown in Figure 1A. Values are the mean ± SEM of the ratios of the number of test (GFP⁺) vs control (YFP⁺) cells measured in 4 replicates for each sample. P values: IK6 vs control. (B) Numbers of CFCs present after 6 weeks in the individual cultures shown in panel A, as enumerated in a 14-day methylcellulose assay and expressed as CFCs per 10³ starting cells. (C) Total nonadherent cells in secondary 6-week cultures (each initiated with one primary culture equivalent). Cells from CP-CML patient #5 showed no growth in secondary cultures. Values are as in panel A. (D) Cumulative cell outputs in serial primary, secondary, and tertiary cultures initiated with cells from CP-CML patients #1 and #4. Cells from CML patients #2 and #3 showed no further growth in tertiary cultures. Values shown are the mean ± SEM. (E) Representative cytospin preparations of nonadherent cells present in tertiary cultures generated from CP-CML #1 (top) and #4 (bottom). (F) Representative flow cytometry plots corresponding to panel E showing analysis of nonadherent cells present in tertiary cultures generated from CP-CML #1 (top) and #4 (bottom). (G) Proportion of apoptotic (PI⁺) IK6- and control-derived CP-CML and normal BM nonadherent cells from 2- to 3-week-old cultures. Values shown are the mean ± SEM from 3 replicate cultures for each of the experiments shown in panel A. P values: IK6 vs control. (H) Proportion of apoptotic (cleaved caspase 3⁺) IK6- and control-derived CP-CML and normal BM CD34⁺ cells in 2- to 3-week-old cultures. Values shown are the mean ± SEM (as indicated) of results for 3 replicate cultures from 4 CP-CML samples and 1 normal BM sample. *P < .05; **P < .01; ***P < .001. NBM, normal bone marrow.