Lineage-specific alterations in residual HSPC by single-cell transcriptomes in patients with AA. (A) Uniform manifold approximation and project (UMAP) visualization of HSPCs based on single-cell transcriptomes (left). Each dot represents a single cell; colors indicate cell clusters with numbered labels (top) and source of donors (bottom) (healthy controls [Ctrls], n = 4; non-SAA, n = 12; SAA, n = 3). Hierarchical clustering showing transcriptional relationships among cell types (right); the dot colors and numbers correspond to their counterparts in the UMAP plot (left). (B) Bar plot showing the ratio of observed to expected numbers of each cell type in patients non-SAA (n = 12). Dots indicate individual patients and dot sizes represent logarithmic transformed P values (χ² test). Error bars represent ± standard error of the mean. (C) Heat-map showing differentially expressed genes (rows) comparing patients with non-SAA (n = 12) with Ctrl across 9 different cell types (columns). Red indicates upregulation in non-SAA, blue indicates downregulation in non-SAA, and yellow indicates no significant change in expression (Wilcoxon rank-sum test, fold change ≥2, Bonferroni adjusted P ≤ .05). (D) Representative expression of selected gene sets illustrating the heterogeneity of transcriptional modulation and differential expression in HSPCs from patients with non-SAA (n = 12) and SAA (n = 3). *P ≤ .05; **P ≤ 0.01; ***P ≤ 0.001 (Wilcoxon rank-sum test). (E) UMAP visualization of HSPC clustering based on regulons. Each pie chart shows the composition of each regulon cluster by cell types (defined based on single-cell gene expression). (F) Representative display of differential regulon activity in hematopoietic stem cells and multipotent progenitors (top) and megakaryocyte/erythroid progenitors (bottom) corresponding to regulon clusters R1 and R3 in (E), respectively. Dark red dots represent cells from SAA (n = 3), dark blue dots represent cells from patients with non-SAA (n = 12), and dark gray dots represent Ctrl cells (left). Blue indicates active regulons and gray represents inactive regulons (right). (G) Heat-map showing regulons that were differentially activated (rows) between patients with non-SAA (n = 12) and Ctrl across 7 different regulon clusters (columns). Blue to red color indicates low to high regulon activity in non-SAA.