Single-cell molecular analysis delineates the heterogeneity of the CML LSC population. (A) Overview of donor cell distribution and BCR-ABL expression in Lin⁻CD34⁺CD38^−/low from CML patients analyzed for single-cell gene expression at diagnosis (CML Dx) (n = 13) and 1 or 3 months following TKI treatment (TKI) (n = 10) compared with nBM. (B) Heat map of normalized gene expression for 71 genes in 2331 Lin⁻CD34⁺CD38⁻ single cells from all donors. Red indicates high expression; blue indicates low expression. Genes are listed to the right and ordered in groups based on which cell state or cell type they characterize, shown to the left. Each row in the heat map corresponds to a specific gene; each column corresponds to a particular single cell. RFC identified 7 subgroups of cells (Myeloid I-IV, Primitive, Lymphoid, and Megakaryocytic/Erythroid [Meg/E]), based on their expression of the predefined gene groups (indicated by white boxes in the heat map). The single asterisk indicates significant upregulated gene expression (P < .001) comparing the cells in the white boxes to all other cells in the heat map. The double asterisk indicates significant upregulated expression comparing the genes in the white box compared with all other genes within the Primitive subpopulation (P < 1 × 10⁻³¹). The row closest to the heat map represents the color coding for each subpopulation where BCR-ABL^pos cells are indicated with dots. The color coding of the second row (sample) indicates donor status (nBM, white; CML Dx, gray; CML patient during TKI treatment [CML TKI], black), and the third row shows the donor distribution (“Patient ID”), where each color represents a donor. (C) Cumulative plots of the time to first division of single CD41⁺CSF2/3R⁻, CD41⁻CSF2/3R⁻, and CSF2/3R⁺ LSCs enriching for subpopulations expressing myeloid, primitive, and Meg/E signatures, respectively. Each plot represents an individual CML patient at diagnosis. Dead cells are excluded. (D) Mean time to first division in the 3 patients analyzed (*P = .002). (E) Lineage distribution of clones derived from single LSCs subdivided by the indicated markers to enrich for subpopulations expressing myeloid, primitive, and Meg/E signatures. The single cells were cultured in stem cell factor, thrombopoietin, interleukin-3 (IL-3), and erythropoietin (condition A) or stem cell factor, thrombopoietin, IL-3, erythropoietin, IL-6, and granulocyte colony-stimulating factor (condition B). The clones were scored by FACS analysis as erythroid (CD235a⁺), myeloid (CD15⁺/CD33⁺), or mixed (CD235a⁺ and CD15⁺/CD33⁺). Morphology analysis confirmed that the CD235a⁺ and CD15⁺/CD33⁺ cells represented erythroid and myeloid cells, respectively. The columns represent pooled data for lineage distribution for 230 single-cell clones from 3 CML Dx patients (condition A) and 164 clones from 2 CML Dx patients (condition B).