Fig 2: Cytokine expression levels in stimulated and freshly isolated human HSPCs. (A) The gating strategy and the population purity after flow cytometry cell sorting. (B) Representative flow cytometry plots showing GM-CSF, IL-6 and TNF-a levels in TLRL-stimulated CD34+ cells from PB or CB. To obtain a sufficient number of HSPCs, lin-CD34+ cells were first expanded in SFEM supplemented with 50 ng/ml SCF. On day 3, these cells were exposed to LPS and Pam3CSK4 for 12 h, and then cells were harvested for flow cytometry analysis (with restimulation II). The data refer to a typical experiment out of three that generated similar results. **P < 0.01, ***P < 0.001; ns, not significant, by Student’s t test. (C) Representative flow cytometry plots showing GM-CSF, IL-6 and TNF-a levels in freshly isolated human blood-derived HSPCs (n = 3 per group). Cells isolated from fresh blood were treated with restimulation II before flow cytometric analysis. The data refer to a typical experiment out of three that generated similar results. **P < 0.01; ns, not significant, by 1-way ANOVA followed by Tukey’s test. The numbers in the flow cytometry plots indicate the proportions of gated cells. APC, allophycocyanin; eF450, eFluor 450; FITC, fluorescein isothiocyanate; NA, not available.

Fig 3: Cytokine production and secretion by PB- or CB-derived HSPCs. (A, B) Adult PB- (A) or neonatal CB-derived (B) CD34+ HSPCs were either left untreated or stimulated with LPS and Pam3CSK4 for 12 h. The percentages of cell expressing GM-CSF, IL-6 and TNF-a were determined by flow cytometry. MFI of the cytokine signals in the corresponding positive cells are shown. The secretion of cytokines in the supernatant was quantified by ELISA, with 20,000 CD34+ cells in 200 µl medium. Data are shown as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001; ns, not significant, by 2-way ANOVA followed by Bonferroni’s test or Student’s t test. (C) The number of cytokine-expressing HSPCs were measured by ELISPOT. CD34+CD38- cells (4,800 cells/well) or CD34+CD38- cells (45,000 cells/well) were incubated for 20 h in ELISPOT plates in the presence or absence of LPS and Pam3CSK4. The data refer to a typical experiment out of three that generated similar results. (D) Summary of two different regulatory modes of cytokine production in HSPCs.

Fig 4: Optimization of cellular restimulation strategy. (A) Schematic representation of the experimental approach. (B) Schematic representation of HSPC isolation and the flow cytometry sorting strategy. (C) Two restimulation strategies, combining pharmacologic stimuli, protein transport inhibitor and culture system, were selected based on the results shown in Supplemental Figures 2–5 . (D, E) Representative flow cytometric analysis of cultured LSK cells exposed to LPS and Pam3CSK4 (D) or Hepa SPSC-SN (E). The intracellular GM-CSF, IL-6 and TNF-a levels were examined. The data refer to a typical experiment out of three that generated similar results. The numbers in the flow cytometry plots indicate the proportions of gated cells. Naïve LSK cells were cultured in the indicated medium for 12 h (D) or 4 days (E) before restimulation. After that, cells were washed and cultured in the restimulation conditions as indicated for an additional 6 h before staining. I, restimulation I; II, restimulation II; Med, control medium (SFEM + SCF); TLRL, control medium supplemented with 100 ng/ml LPS and 1 µg/ml Pam3CSK4; Hepa, control medium supplemented with 5% (v/v) Hepa mice-derived SPSC-SN; BV421, Brilliant Violet 421; PE, phycoerythrin.