Unphosphorylated STAT5 constrains HSC differentiation and upregulates transcriptional programs associated with HSC maintenance. (A) Schematic diagram showing the experimental outline of the ex vivo functional analysis of STAT5-deficient or WT ESLAM (CD45⁺CD150⁺CD48⁻EPCR⁺) HSCs that were transduced with lentivirus containing STAT5B-YF or EV in maintenance cultures.⁶² After 3 days of transduction, GFP⁺ living cells were FACS sorted into single-cell assays. (B) Bar plots showing the proportion of cells expressing mature lineage markers (Ter119⁺/Ly6g⁺/CD11b⁺/B220⁺/CD3e⁺). Each dot represents a single clone and bars represent the mean lineage-positive marker frequency (±SEM). Asterisks indicate significant differences as determined by Student t tests (∗∗∗P < .001; ∗∗P < .01). The results are from 6 to 5 independent biological replicates across 5 experiments in STAT5^+/+ settings and 4 to 3 independent biological replicates across 3 experiments in STAT5^−/− settings. (C) Schematic diagram showing the outline of the scRNAseq analysis of WT ESLAM HSCs that were transduced with lentivirus containing STAT5B-YF or EV in maintenance cultures⁶² and that were allowed to expand for 5 days. GFP⁺ living cells were then sorted for 10X Genomics scRNAseq. (D) Bar plots showing the proportion of annotated cell types in GFP⁺ HSC-derived cultures after 5 days in SCF/IL-11 cultures; single cells were projected onto a previously published scRNAseq data set of LK HSPC cells⁶⁰ and then onto a phenotypically-defined HSPC data set,⁶¹ and cell types were annotated based on their nearest neighbors to ascribe cell identity and cell type annotation. The results are from 2 independent biological replicates in 2 experiments. (E) Gene set enrichment plot showing that STAT5-YF–infected, transcriptionally defined LT-HSCs (n = 83) are depleted in a DNA replication gene signature when compared with EV-infected LT-HSCs (n = 53). The NES (−5.24) and FDR (<0.001) are indicated. (F) Left: bar plots showing the cell cycle phase frequency of ESLAM HSC-derived cultures infected with either EV (n = 5) or STAT5-YF (n = 4) lentivirus after 5 days in maintenance culture media.⁶² The cell cycle status was derived from Ki67/DAPI staining (right). G₀ represents quiescent cells that are Ki67^lowDAPI^low; G₁ represents cells in the early growth phase, which are Ki67^highDAPI^low; S-G2-M represents cells in DNA synthesis, late growth, and mitosis stages of active cell cycling and are Ki67^highDAPI^high. (G) Violin plot showing the geometric mean distribution of HSC scores in LT-HSCs expressing STAT5B-YF or EV. The HSC score was calculated using the HSC score tool that identifies potential mouse BM HSCs from scRNAseq data.⁶³ This tool considers the expression of genes that are either positively or negatively corelated with HSC long-term repopulating capacity.⁶⁴ (H) Violin plots showing significantly differentially expressed genes that are positively associated with functional long-term repopulating HSCs (Pdzk1ip1, Mettl7a1, Mllt3, and Gimap1), negatively associated with functional long-term repopulating HSCs (Serpinb1a and Hsp90aa1), or genes with reported functions in maintaining HSCs (Hlf, Chd9, Pbx1, and Plxnc1). All data were combined from 2 independent experiments. Asterisks indicate significant differences as determined by Student t tests (∗∗∗∗P < .0001; ∗∗∗P < .001; ∗∗P < .01; ∗P < .05). GMP, granulocyte-macrophage progenitor; LMPP, lymphoid-myeloid progenitor; MEP, Meg/Ery progenitors; ns, not significant.