Igf2bp2 expression in young HSCs cosegregates with Lin28b, Igf/Pi3k, and stemness-related gene expression signatures. Freshly purified myeloid-biased HSCs (CD150^highCD34⁻LSK) from 6-week-old male wild-type mice were analyzed by scRNA-seq (single-pool analysis of n = 5 mice). (A) UMAP plot with Seurat clustering analysis revealing 9 distinct clusters (0-8). (B-I) Feature plots and histograms on target genes of the Lin28/Igf2bp2 pathway depicting the expression levels and the percentage of positive cells in the HSC subclusters for Igf2bp2 (B-C), Lin28b (D-E), Igf2 (F-G), and Igf1 (H-I). Gray dots indicate no expression, and the intensity of the red dots indicates the expression level of each gene. (J) Heat map of DEGs in myeloid-biased HSCs from cluster 3 (enriched for Igf2bp2 expressing cells) compared with myeloid-biased HSCs from clusters 0, 4, 5, and 8 (Bonferroni-adjusted P < .01). The color scale indicates gene expression level. The cluster is enriched for expression of known stemness-related genes (Mllt3, Cdkn1c, Procr, Xbp1, and Slfn2); for imprinted genes expressed in quiescent-enriched, long-term HSCs³³ (Meg3, Rian, and Cdkn1c); and for positive regulators of Igf/Akt signaling (Pik3r1 and Igf1), but is depleted for the expression of inhibitors of Igf/Akt signaling (Cmtm7 and Igfbp4). (K) Heat map of the expression pattern of marker DEGs of myeloid-biased HSCs from cluster 3 in panel J that overlapped with the DEGs in young Igf2bp2^−/− myeloid-biased HSCs vs Igf2bp2^+/+ myeloid-biased HSCs from bulk RNA sequencing analysis (Figure 2). UMAP, Uniform Manifold Approximation and Projection.