STAT5-deficient HSCs display reduced cell cycle entry, increased differentiation, and reduced retention of lineage-negative progeny. (A) Gene set enrichment analysis (GSEA) plots showing depleted cell cycle–related signatures in STAT5-deficient ESLAM (CD45+CD150+CD48−EPCR+) HSCs. scRNAseq analysis using the Smart-seq platform was performed on FACS-isolated ESLAM HSCs from STAT5f/f Cre- or STAT5f/f Cre+ BM; 132 STAT5-deficient and 132 WT HSCs passed quality control and were used for downstream analysis. The normalized enrichment scores (NES) and false discovery rate (FDR) are indicated. (B) Plots showing the cell cycle scores of transcriptionally defined LT-HSCs, ST-HSCs, and MPPs that were isolated from scRNAseq data sets of WT and STAT5-deficient BM LK cells (STAT5 WT, n = 3; STAT5KO, n = 3). (C) Line graphs showing the proportion of ESLAM HSCs that past first, second, third, and fourth divisions at given timepoints (y-axis) in the single cell in vitro analysis (mean ± SEM). The results are from 3 biological replicates across 3 experiments. (D) Bar plots (left) showing the mean fluorescent intensity (MFI) of pSTAT5 antibody staining of ESLAM HSCs by intracellular flow-cytometry analysis in unstimulated maintenance culture conditions62 (SCF/IL-11) or TPO (200 ng/mL) positive control conditions (mean ± SEM). Right; representative histogram of the intracellular flow-cytometry analysis showing the intensity of pSTAT5 staining in each condition. The results are from 3 biological replicates. (E) Bar plot showing the number of cells in each well at day 5 from an initial culture of 50 ESLAM HSCs in SCF/IL11 maintenance conditions. The number of cells that expressed mature lineage markers (Ter119+, Ly6g+, CD11b+, NK1.1+, B220+, CD19+, or CD3e+) and the number of lineage-negative cells are shaded in different colours (mean ± SEM). The results are from 9 to 7 biological replicates across 4 experiments. (F) Bar plot showing the proportion of cells that expressed mature lineage markers after 5 days in culture that originated from 50 ESLAM HSCs (mean ± SEM). (G) Bar plot showing the proportion of cells that expressed specific mature lineage markers for monocytes (Mons) and granulocytes (Grans; Ly6g+), Grans and macrophages (MacsCD11b+), erythroid (Ery; Ter119+), lymphocytes (LYMs; CD3e+/CD19+/B220+), and natural killer cells (NK; NK1.1+) after 5 days in culture that originated from 50 ESLAM HSCs (mean ± SEM). (H) Bar plot showing the frequency of Ter119+ cells after 5 days in culture that originated from 50 ESLAM HSCs (mean ± SEM). Asterisks indicate significant differences as determined by Student t test (∗∗∗∗P < .0001; ∗∗∗P < .001; ∗∗P < .01; ∗P < .05). KO, knockout.

STAT5-deficient HSCs display reduced cell cycle entry, increased differentiation, and reduced retention of lineage-negative progeny. (A) Gene set enrichment analysis (GSEA) plots showing depleted cell cycle–related signatures in STAT5-deficient ESLAM (CD45+CD150+CD48−EPCR+) HSCs. scRNAseq analysis using the Smart-seq platform was performed on FACS-isolated ESLAM HSCs from STAT5f/f Cre- or STAT5f/f Cre+ BM; 132 STAT5-deficient and 132 WT HSCs passed quality control and were used for downstream analysis. The normalized enrichment scores (NES) and false discovery rate (FDR) are indicated. (B) Plots showing the cell cycle scores of transcriptionally defined LT-HSCs, ST-HSCs, and MPPs that were isolated from scRNAseq data sets of WT and STAT5-deficient BM LK cells (STAT5 WT, n = 3; STAT5KO, n = 3). (C) Line graphs showing the proportion of ESLAM HSCs that past first, second, third, and fourth divisions at given timepoints (y-axis) in the single cell in vitro analysis (mean ± SEM). The results are from 3 biological replicates across 3 experiments. (D) Bar plots (left) showing the mean fluorescent intensity (MFI) of pSTAT5 antibody staining of ESLAM HSCs by intracellular flow-cytometry analysis in unstimulated maintenance culture conditions62 (SCF/IL-11) or TPO (200 ng/mL) positive control conditions (mean ± SEM). Right; representative histogram of the intracellular flow-cytometry analysis showing the intensity of pSTAT5 staining in each condition. The results are from 3 biological replicates. (E) Bar plot showing the number of cells in each well at day 5 from an initial culture of 50 ESLAM HSCs in SCF/IL11 maintenance conditions. The number of cells that expressed mature lineage markers (Ter119+, Ly6g+, CD11b+, NK1.1+, B220+, CD19+, or CD3e+) and the number of lineage-negative cells are shaded in different colours (mean ± SEM). The results are from 9 to 7 biological replicates across 4 experiments. (F) Bar plot showing the proportion of cells that expressed mature lineage markers after 5 days in culture that originated from 50 ESLAM HSCs (mean ± SEM). (G) Bar plot showing the proportion of cells that expressed specific mature lineage markers for monocytes (Mons) and granulocytes (Grans; Ly6g+), Grans and macrophages (MacsCD11b+), erythroid (Ery; Ter119+), lymphocytes (LYMs; CD3e+/CD19+/B220+), and natural killer cells (NK; NK1.1+) after 5 days in culture that originated from 50 ESLAM HSCs (mean ± SEM). (H) Bar plot showing the frequency of Ter119+ cells after 5 days in culture that originated from 50 ESLAM HSCs (mean ± SEM). Asterisks indicate significant differences as determined by Student t test (∗∗∗∗P < .0001; ∗∗∗P < .001; ∗∗P < .01; ∗P < .05). KO, knockout.

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