Ruxolitinib (RUX) enhances HSPC clonogenicity and maintains transplantable HSCs. (A) Schematic diagram showing intracellular flow cytometric analysis of STAT5 proteins in RUX-treated WT ESLAM HSCs (CD45⁺CD150⁺CD48⁻EPCR⁺). WT ESLAM HSCs were sorted into serum–starved media and starved for 1 hour before a 30-minute stimulation with complete medium containing IL-3, IL-6, and SCF in the presence or absence of RUX,⁶⁷ and a stimulation cocktail containing thrombopoietin (THPO), Flt3-L, and interferon alfa for positive control was included. Cells were then fixed and stained for intracellular flow cytometry. (B) Bar plots showing the MFI of pSTAT5 antibody staining in ESLAM HSCs, described in panel A, normalized to the unstimulated condition, which is indicated with the dotted line (mean ± SEM). Each dot represents the MFI of ESLAMs from a single mouse. The results are from 3 independent experiments. (C) Bar plots showing the MFI of total-STAT5 (tSTAT5) antibody staining in ESLAM HSCs, described in panel A, normalized to STAT5-deficient HSPCs, which is indicated with the dotted line (mean ± SEM). The results are from 3 independent experiments. (D) Bar plots showing cell number per well in HSC-derived cultures at each dose of RUX or vehicle after 7 days (mean ± SEM). A total of 50 ESLAMs were seeded per well in 96-well plates in IL-3/IL-6/SCF⁶⁷ cultures and were treated with DMSO or the indicated doses of RUX. The results are from 6 independent experiments. (E) Bar plot showing the proportion of cells that expressed lineage-positive markers (Ter119⁺/Ly6g⁺/CD11b⁺/B220⁺/CD3e⁺) after 7 days in culture at different concentrations (nM) of RUX (mean ± SEM). A total of 50 ESLAMs were seeded per well in 96-well plates in IL-3/IL-6/SCF⁶⁷ cultures and were treated with the indicated doses of RX. The results are from 6 independent experiments. (F) Bar plots showing the clone survival rate of single HSCs after 5 days in culture. Single ESLAM HSCs were sorted per well and treated with vehicle or RUX. Clone survival rate was the proportion of wells that contained cells at day 5. Each dot represents the frequency of surviving clones from each of 3 independent experiments; the bars show the mean ± SEM. (G) Schematic diagram showing the serial colony replating assays and in vivo functional analysis for ESLAM HSCs treated with RUX or vehicle. A total of 50 WT ESLAM HSCs were sorted per well into complete media⁶⁷ with scaled doses of RUX or vehicle. Cells were harvested after 7 days and plated into serial colony replating assays. ESLAM HSC (CD45.2⁺)–derived cells after 5 days in culture were harvested and transplanted into lethally irradiated recipient mice (CD45.1⁺) with 3 × 10⁵ fresh BMMNCs from competitor mice (CD45.1⁺/CD45.2⁺). Blood was analyzed every 28 days for 6 months. Secondary transplants were then set up by transplanting 3 × 10⁶ BM cells from the primary transplant recipients. (H) Bar plots showing the number of colonies produced by HSC-derived cultures treated with vehicle or RUX (250 or 1000 nM) for 7 days, normalized to the number of colonies produced by vehicle-treated cultures at each week of replating. The results are shown as mean ± SEM and were from 5 independent experiments, 3 of which included 1000 nM. Asterisks indicate significant differences as determined by Mann-Whitney U tests (∗∗P < .01; ∗P < .05). (I) Scatter dot plot with linear regression line of best fit showing the peripheral blood donor chimerism in primary (left) and secondary (right) recipients transplanted with 5-day ex vivo cultured HSCs with RUX or vehicle. A total of 50 ESLAMs from WT mice were seeded per well in IL-3/IL-6/SCF culture conditions and given DMSO or 250 nM of RUX for 5 days before the cells were harvested and pooled for each condition, and an equivalent of 10 starting ESLAMs was transplanted per recipient with 3 × 10⁵ competitor BM cells. Each dot indicates the mean donor chimerism and are shown as mean ± SEM. Black asterisks indicate significant differences in the slopes of the linear regression modeling that compared chimerism of RUX-treated donor cell with DMSO-treated donor cell chimerism in the primary recipients (∗∗P < .01). Blue asterisks indicate significant differences in the y-intercepts of linear regressions modeling that compared chimerisms of RUX-treated donor cells with DMSO-treated donor cells in secondary transplants (∗∗∗P < .001). (J) Bar plots showing the donor chimerism within the ESLAM HSC compartment at the end of primary and secondary recipients of 5-day ex vivo cultured HSCs with RUX or vehicle. The data are shown as the mean ± SEM. (K) Violin plot showing the geometric mean distribution of HSC scores in LT-HSCs from the 10x scRNAseq data set of the cells treated with RUX or DMSO. The scores were 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 correlated with HSC long-term repopulating capacity.⁶⁴ (L) Violin plots showing significantly differentially expressed genes that are positively associated with functional long-term repopulating HSCs (Pdzk1ip1, Mettl7a1, Mllt3, and Gimap6), negatively associated with functional long-term repopulating HSCs (Hsp90aa1 and Cdk6), or genes with reported functions in maintaining HSCs (Hlf, Pbx1, Chd9, 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) unless otherwise indicated. ns, not significant.