Figure 4.

NA10HD markedly enhances the expansion of normal and Ph⁺/BCR-ABL⁺ LTC-ICs in vitro in LTCs and has the greatest effect on the more primitive CD34⁺CD38⁻ cells. (a) Experimental design (using cells from the same four CP-CML patients studied in Figure 3). Assays were performed on cells that had been maintained in GF-SFM suspension cultures for 3 days after exposure to virus. (b) Data for control-transduced cells are shown in open symbols/bars and for NA10HD-transduced cells are shown in black symbols/bars. Comparison of the absolute number of LTC-derived GFP⁺ BCR-ABL⁺ CFCs found in primary (wk 6) and secondary (wk 12) LTC-IC assays normalized to day 0 values (pretransduction values, dotted lines) obtained from the same number of starting CD34⁺ cells from the same patient. Arrows indicate values below the detection limit corresponding to the data point. (c) The same data for the LTC-IC-derived GFP⁺ normal (BCR-ABL⁻) CFCs are shown separately for patient 4 (as no BCR-ABL⁺ CFCs were found in LTCs initiated with these cells). (d) Comparison of BCR-ABL transcript levels as a function of NA10HD transcript levels (quantified relative to GAPDH transcripts) in extracts of individual colonies produced from CFCs generated in 6-week LTCs initiated with transduced cells from patients 1, 2 and 3. Results are expressed as 2^{−(Ct BCR−ABL−Ct GAPDH)} for BCR-ABL transcripts and 2^{−(Ct NA10HD−Ct GAPDH)} for each colony (R²=0.0007; Pearson test showing no significant correlation, P=0.87). (e) Comparison of the effect of NA10HD on the CD38⁺ and CD38⁻ subsets of CD34⁺ CML cells (patient 2). Data shown are the absolute number of LTC-derived GFP⁺ BCR-ABL⁺ CFCs produced in primary (wk 6) and secondary (wk 12) LTC-IC assays expressed per 10⁵ starting CD34⁺CD38⁺ cells or CD34⁺CD38⁻ cells. (f) The same experimental design was followed as in panel (e) but the CD34⁺CD38⁻ cells were additionally subdivided into a Rho⁺ and Rho⁻ fraction (cells from patient 1). Arrows show detection limits. ND, not done.