cHSPCs show a preactivated state associated with higher in vitro differentiation efficiency than BM-HSPCs. (A) Tile plot of the top 5 GO-BP macrocategories for each cluster. Statistically significant GO-BP ontology gene sets (adjusted P < .05) with an enriched expression in PB (NES > 0) or BM (NES < 0) are shown in blue and red, respectively. For each macrocategory, color intensity is proportional to NES absolute values. Macrocategories were classified in diverse groups, according to the associated biological functions. (B) Heat map showing the scaled expression of cluster 1 marker genes that are differentially expressed between PB and BM (adjusted P < .05; logFC ≥0.4 or ≤0.4.) cells. Genes are grouped by biological functions. Annotation for BM (red) and PB (blue) cells is reported. (C) UMAP embedding grouping cells by source and coloring by inferred cell cycle. (D) Histograms representing the percentage of cells in G0, G1, S, and G2M cell cycle phases for each cluster in the BM (top) and the PB (bottom) data sets. The x-axis shows cluster annotation according to the HSPC subset transcriptional signatures. Classification of the cell cycle activity was performed on the overall data set, shown in supplemental Figure 7B, and then reported on BM (top) and PB (bottom) single data sets. (E) Summary of the differentiation efficiency of PB-HSPCs (n = 4) and BM-HSPCs (n = 3) in our single-cell in vitro differentiation assay. The number of seeded wells, the count of wells showing differentiated progeny after 3 weeks of culture, and the total differentiation efficiency starting from one single cell are reported. (F) Pie charts representing the frequencies of BM- and PB-HSPC showing specific lineage scores detected at the end of the single-cell in vitro differentiation assay. GO-BP, gene ontology-biological processes; NES, normalized enrichment score.