Clonogenic growth and in vivo repopulation of CML SCs. (A) Left: Sorted (>98% pure) CD34⁺/CD38⁻/CD26⁻ SC, CD34⁺/CD38⁻/CD26⁺ SC, and CD34⁺/CD38⁺ sorted progenitor cells (CP CML) were plated in methylcellulose and incubated at 37°C for 16 days. Then CFU-GM–derived colonies were counted under an inverted microscope (black bars). Thereafter, colonies were picked and BCR/ABL1 mRNA expression was determined by qPCR (white bars). Results represent the mean ± SD of triplicates. Right: Purified CD34⁺/CD38⁻/CD26⁻, CD34⁺/CD38⁻/CD26⁺, and CD34⁺/CD38⁺ cells were incubated on irradiated M2-10B4 feeder cells for 3 weeks. Then cells were harvested and cultured in a CFU-GM assay. CFU-GM colonies (black bars) were counted by microscopy and BCR/ABL1 mRNA levels were determined by qPCR (white bars). (B) Experimental setup of xenograft experiments with purified CML cells. First, CML cells (CP) were depleted from lineage⁺ cells and separated into CD26⁺ cells and CD26⁻ cells by cell sorting (purity >98%). The 2 fractions were separately injected intravenously into irradiated NSG mice (0.1-0.8 × 10⁶ CD26⁺ cells per mouse and 0.5-1.5 × 10⁶ CD26⁻ cells per mouse). (C) After 16 weeks or 28 weeks, flushed BM cells of NSG mice injected with CD26⁻ cells (blue symbols) or CD26⁺ cells (red symbols) were analyzed for the presence of CD33⁺ myeloid cells (circles) and CD19⁺ B cells (triangles) by flow cytometry. (D) Flow cytometry patterns of engrafted cells in 2 representative mice, one injected with CD26⁻ SC, where multilineage BCR/ABL1^– engraftment was found (upper panel) and one with CD26⁺ SC, where engraftment with BCR/ABL1⁺ myeloid cells was seen (lower subpanel). The table shows a summary of engraftment results with percentages of human CD45⁺ cells, CD33⁺ cells, and CD19⁺ cells, and the detection of BCR/ABL1 by qPCR. n.d., not determined.