Figure 3.

Forced expression of NA10HD in CD34⁺ cells from CML patients promotes a rapid and sustained increase in LTC-ICs without effects on their ability to differentiate. (a) Experimental design used to assess the effect of NA10HD on LTC-IC activity in cultures of CD34⁺ cells isolated from four CP-CML patient's samples that initially contained variable numbers of Ph⁺/BCR-ABL⁺ LTC-IC assays were performed 2 and 7 days post-transduction (day 3 and 8 in GF-SFM suspension cultures). (b) Data for control-transduced cells are shown as open symbols/bars and data for NA10HD-transduced cells are shown as black symbols/bars. Comparison of total GFP⁺ cell numbers in 6-week-old LTCs initiated with NA10HD- or control-transduced cells (left panel) and time course data from one experiment (patient 2). (c) Comparison of the number of GFP⁺ CFCs in the 6-week-old LTCs initiated with NA10HD- or control-transduced cells in the suspension cultures assessed 2 and 7 days after exposure to virus. All values were normalized to the day 0 value for each patient. Arrows indicate values below the detection limit corresponding to the data point. (d) Giemsa staining of cells (mainly macrophages) found in the non-adherent fraction of 5-week-old LTCs initiated with control-transduced cells from patient 2 as compared with the non-adherent fraction of corresponding LTCs initiated with NA10HD-transduced cells found to contain mainly granulocytes. Scale bar: 50 μm. (e) Photomicrographs of colonies produced in methylcellulose assays of LTC-derived CFCs derived from both control- and NA10HD-transduced CML cells illustrating their normal appearance. Scale bar: 500 μm. (f) Representative FACS profiles of immuno-stained cells harvested from CFC assays of 6-week LTCs initiated with control- or NA10HD-transduced cells showing their similar content of mature erythroid (GlyA⁺CD33⁻), immature (CD33⁺CD15⁺) and mature (CD33⁻CD15⁺) granulocytes, and monocytes/macrophages (CD33⁺CD14⁺).