The most TKI-insensitive Primitive subpopulation can be defined as Lin⁻CD34⁺CD38^−/lowCD45RA⁻cKIT⁻CD26⁺. Immunophenotypic characterization of sorted Lin⁻CD34⁺CD38^−/low single cells at diagnosis (A) and at TKI treatment (B). The left panels depict representative FACS plots for cell surface expression of the indicated marker on individual BCR-ABL^pos cells, color coded according to their subpopulation defined by the RFC of the single-cell gene expression data. The dark gray dots represent BCR-ABL^neg cells, the lighter gray dots are unsorted but FACS recorded cells. The right panels show pooled data from 3 patients and the proportion of BCR-ABL^pos cells in each subpopulations that express the indicated marker on their cell surface. (C) FACS analysis of Lin⁻CD34⁺CD38^−/low with a combination of markers for prospective isolation of TKI-persistent CML LSCs. The FACS plots exemplify 1 representative CML patient following TKI treatment. The gates are set according to the isotype control antibody for CD26, IL1RAP, cKIT, and CD25, respectively. The bar charts depicts pooled data from 3 individual patients following 3 months of TKI treatment and display the relative yield and purity of total BCR-ABL^pos cells (green) and of the subpopulation with Primitive molecular program (purple) that is achieved when using the Lin⁻CD34⁺CD38^−/low protocol alone (-) or the indicated combination of markers.