Fig 2: Droplet-based single-cell assays for transposase-accessible chromatin sequencing (scATAC-seq) analysis of thymic epithelial cells (TECs) in 4-week mice.(A) Uniform manifold approximation and production (UMAP) plot of scATAC-seq data from TECs (EpCAM+ CD45– TER119–) from 4-week mice. Cell clusters are separated by colors and numbers in the plot. The two datasets were integrated using the Seurat package. The graph on the right shows percentages of each cluster in the total number of cells detected (15,255 cells). (B) Chromatin accessibility of typical marker genes of TECs. Accessibility in each gene region is represented in red. (C) Violin plot depicting chromatin accessibility in Aire and Cd80 gene regions in each cluster. (D) Pseudo-bulk accessibility tracks of the Aire gene region in each cluster (upper panels) and frequency of sequenced fragments within the Aire gene region of individual cells in cluster 0, 2, and 4 (lower panels).

Fig 3: Droplet-based single-cell RNA sequencing (scRNA-seq) analysis of thymic epithelial cells (TECs) in 4-week mice.(A) Uniform manifold approximation and production (UMAP) plot of scRNA-seq data from TECs (EpCAM+ CD45– TER119–) from 4-week mice. Cell clusters (R0 to R17) are indicated by colors and numbers in the plot. The graph on the right shows the percentages of each cluster in the total number of cells detected (11,792 cells). (B) Violin plots depicting expression levels of typical TEC marker genes in each cluster.

Fig 4: AML-Derived Alteration of the BM Vascular Architecture and Function(A) Quantification of CD31+ ECs in the BM (shown as percentage of CD45-Ter119- BM cells) of non-transplanted mice (ctrl) and mice transplanted with HSPCs (CB) or AML patient-derived samples, as depicted. Each dot represents an individual mouse. Ctrl, n = 22; CB, n = 29; AML patients (AML1, 2, 3, 5, 6, 7, 8, 9), n = 30. Data are shown as mean ± SEM.(B) Absolute number of CD31+ ECs in the BM (2 femurs, 2 tibias, and 2 iliac crests) of non-transplanted mice (ctrl) and mice transplanted with CB-derived HSPCs (CB) or AML patient-derived samples, as depicted. Each dot represents an individual mouse. Ctrl, n = 17; CB, n = 26; AML patients (AML1, 2, 6, 7, 8, 9), n = 23. Data are shown as mean ± SEM.(C) Frequency of Sca-1high and Sca-1low CD31+ cells in the BM of non-transplanted mice (ctrl) and mice transplanted with AML patient-derived samples. Ctrl, n = 19; AML patients (AML 2, 3, 6, 8, 9), n = 16. Data are shown as mean ± SEM.(D) Representative 3D reconstruction of BM vasculature of the calvarium of non-transplanted mice (ctrl) and mice transplanted with CB-derived HSPCs (CB) or AML patient-derived samples, as depicted, imaged via 2P microscopy 1 min after injection of 655-conjugated NT-Qtracker as vessel-pooling agent. White arrows pointing at sinusoids. Data are representative of triplicates in 4 independent experiments. Scale bars represent 70 µm.(E) Quantification of vascular mean diameter in the calvarium BM of non-transplanted mice (ctrl) and mice transplanted with CB-derived HSPCs (CB) or human AML-derived samples, as depicted, using IMARIS filament tool. Dots represent the diameter of vascular fragments in the z stack of the calvarium of at least 3 mice per group. Ctrl, n = 235; CB, n = 232; HL60, n = 91; ML1, n = 148; U937, n = 85; AML6, n = 130; AML8, n = 94. Red lines represent the mean ± SEM.(F) Representative 3D reconstruction of BM hypoxia imaged via intravital microscopy using the HypoxiSense probe together with vasculature (dextran) and Nestin+ cells in non-transplanted mice or mice transplanted with AML6 patient-derived cells, as depicted. Scale bars represent 50 µm.(G) Distribution and relative frequency of vessel distances to hypoxic areas in the BM of non-transplanted mice (ctrl) or mice transplanted with AML6 patient-derived cells, as depicted.ns, not significant; *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S1.

Fig 5: Normoxia changes the transcriptional output to suppress osteogenesis ASchematic representation demonstrating the isolation protocol and the culture conditions of bone MSCs isolated from the back limbs of young (~3–5 months old) mice. After collection of the limbs, clean bones were cut into small pieces, which were then treated with collagenase for 1 h at 37°C. Cells and bone fragments were seeded in flasks and incubated for 10 days under 2% O2. On day 10, cell sorting was performed using flow cytometry and selecting the CD45-/Ter-119-/Sca-1+/CD140a+ mesenchymal stem cell population. The isolated population was then split into two groups: one was transferred back to hypoxia, whereas the other one was shifted to normoxia. Cells were cultured under these conditions for 7 days.B, CRepresentative images (B) and quantification (C) of Oil Red O staining of hypoxia- and normoxia-cultured cells, 9 days after induction of adipogenesis. n = 5 biologically independent replicates. Results are shown as mean ± SEM and statistical significance was determined using a two-sided unpaired t-test.D, ERepresentative images (D) and quantification (E) of Alizarin Red S staining of hypoxic, normoxic and reversed hypoxic (R_2% O2) cells, 12 days after induction of osteogenesis. Cells were exposed to 21% O2 for 7 days and then moved back to 2% O2, where osteogenesis was induced after 4 days. n = 3 for hypoxic cells and n = 5 biologically independent experiments for normoxic and reversed-hypoxic cells, and merged results are shown in (E). Results are shown as mean ± SEM and statistical significance was determined with ordinary one-way ANOVA, using Holm–Sidak's multiple-comparisons test.FPrincipal component analysis (PCA) plot showing clustering of hypoxia- and normoxia-cultured cells after RNA-seq. n = 4 biologically independent replicates.GGO enrichment analysis for down-regulated genes upon exposure to normoxia, as identified by RNA-seq. Data information: Scale bars, 500 µm. Source data are available online for this figure.