Fig. 6

Molecular and functional differences between CD49f⁺ Subset2, Subset2 cells, LMPPs and MLPs. a–c Single-cell RNA-seq analysis of 49f⁺ Subset2 (n = 119), Subset2 (n = 100), LMPP (n = 140) and MLP (n = 134) single cells. a Left: 3D tSNE representation of indicated populations performed on highly variable genes (5831 genes). Right: density plot of the distribution of indicated populations along the first tSNE component. p < 0.01 by Kruskal–Wallis test with multiple comparison. b Number of significant differentially expressed genes between LMPP and the indicated populations (FDR < 0.05 by DESeq2). c Log₁₀ normalised expression of selected genes differentially expressed both between 49f⁺ Subset2 and LMPP and Subset2 and LMPP in single cells from indicated populations. d Cell surface expression of KIT on Subset2 and LMPP cells. Left: representative flow cytometry histogram, Right: quantification (n = 3 independent CBs, *p < 0.05 and **p < 0.01 by one-way ANOVA with Tukey’s multiple comparison). e–g In vitro differentiation assay of Subset2 and LMPP single cells (n = 240 single cells from two independent CBs plated per population) in conditions supporting differentiation of My, B and NK cells. e Clonogenic efficiency of Subset2 and LMPP cells (n = 2 independent CBs). f Percentage of colonies of the indicated type derived from Subset 2 (n = 153 colonies from two independent CBs) and LMPP (n = 72 colonies from two independent CBs) single cells, *p < 0.05 by two-tailed paired t-test. d–f Shown is mean ± SEM. g Boxplots of the size of colonies generated by Subset2 (n = 153 colonies from two independent CBs) and LMPP (n = 72 colonies from two independent CBs) single cells, **p < 0.01 by two-tailed Mann–Whitney test. Boxplots show median, interquartile and range. h Graphical representation of the single-cell structure of the 49f⁺ HSC compartment proposed here