Fig 2: BMSC-Dependent GSH-Related Pathways Protect AML Cells from Chemotherapy(A) Reduced glutathione (GSH) measured by mBCI staining in AML blasts and BMSCs cultured alone or together for 24 h in presence of AraC.(B) Frequency of AraC-treated GSHlo/hi AML cells monocultured (upper bar) or cocultured with BMSCs (middle and lower bar), separating leukemic blasts, which contain mitochondria derived from previously labeled BMSCs (lower bar) from those containing only blasts’ mitochondria (middle and upper bars); n = 4–5.(C) mRNA expression of Gpx1 and Gpx3 in AML blasts and BMSCs cultured alone or together for 24 h in presence of AraC.(D) GSH measured by mBCI staining in LKlo WT or iMLL-AF9 cells from mice with/without nestin+ cell depletion (Nes-creERT2;iDTA and control littermates) treated as in Figure S3H. Mice were treated with standard chemotherapy (AraC+) or vehicle (AraC-). (A, C, and D) Each dot represents a biological replicate. Data represent mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. Unpaired two-tailed t test. (B and D) One-way ANOVA followed by pairwise Bonferroni comparisons.

Fig 3: Unlike Bulk BM Stromal Cells, Nestin+ Niche Cells Are Preserved in Human and Murine AML and Promote Leukemogenesis(A–D) Representative examples of immunohistochemistry for human NESTIN (brown) and human CD34 (pink) in BM samples from AML patients. CD34- AML (A), CD34+ AML ([B] and [C]), and MLL-AF9+ AML (D) show increased NESTIN+ niches (arrows) and CD34+ vessels (arrowheads). Scale bar, 10 µm (A–C), 100 µm (D).(E) Quantification of NESTIN+ niches from (A and B) (control n = 12; MLL-AF9- AML n = 56; MLL-AF9+ AML n = 5). *p < 0.05, **p < 0.01, one-way ANOVA and Bonferroni comparisons.(F) Scheme showing the induction of AML in primary, non-transplanted Nes-GFP mice to study BMSC changes during leukemogenesis.(G) Fold change in the number of BM stromal cells (CD45- Ter119-CD31-) and BMSCs expressing low or high levels of Nes-GFP (NesGFPlow/high) in the BM of control (C) Nes-GFP;rtTA mice and Nes-GFP;rtTA;iMLL-AF9 (AML) mice. Numbers were normalized with the average of WT controls in each independent experiment. Mice were analyzed 8–10 weeks after inducing MLL-AF9 expression. Dots represent data from individual mice (n = 2 independent experiments). Data are mean ± SEM. Unpaired two-tailed t test.(H) Scheme showing experimental depletion of nestin+ cells in primary, non-transplanted leukemic mice. MLL-AF9 expression (iMLL-AF9) is induced with doxycycline.(I) Nestin+ cell depletion extends AML mouse survival. Kaplan-Meier survival curve of primary iMLL-AF9 mice in control group (black, n = 9) or after nestin+ cell depletion (red, n = 11). Logrank test.

Fig 4: BMSCs Provide Leukemic Blasts with Antioxidant Defense during Chemotherapy(A and B) ROS measured by DH123 staining (A) and lipid peroxidation detected with BODIPY581/591 (B) in AML blasts and BMSCs cultured alone or together for 24 h in presence of AraC. Each dot is a biological replicate. **p < 0.05; unpaired two-tailed t test.(C) Selected pathways from the GO enrichment analysis of significantly upregulated mRNA (RNA-seq) of CD45-CD31-Ter119-Nes-GFP+ cells isolated from the BM of primary iMLL-AF9 mice or control mice. The GO term, adjusted p value and differentially expressed genes in each category are indicated.(D) Selected pathways from the GSEA analysis of RNA-seq of hematopoietic-lineage-negative ckitlow (LKlo) cells isolated from the BM of AML mice with or without nestin+ cell depletion. Lethally irradiated CD45.2 control mice or Nes-creERT2;iDTA mice were transplanted with 106 iAML (rtTA;MLL-AF9) CD45.2+ BM nucleated cells and 106 CD45.1+ WT BM nucleated cells. Doxycycline administration started 2 weeks after transplant; tamoxifen was administered 4 weeks post-transplant; and mice were sacrificed and analyzed 4 weeks later. BM MLL-AF9+ lin- ckitlow (LKlo) cells were sorted from leukemic mice with or without nestin+ cell depletion. The pathway term, signature, and adjusted p value are indicated.(E) Heatmap showing antioxidant and GSH related among the top upregulated (red) protein pathways detected by proteomics in AraC-treated AML blasts cultured alone or cocultured with mesenspheres for 24 h. Quantitative proteomics results were analyzed using the WSPP statistical model (Navarro et al., 2014) and expressed in log2 fold change values in units of SD (Zq) with respect to baseline. Significant protein changes (adjusted p < 0.05) are indicated; two-sided Fisher test, 3 biological replicates in each condition.(F) SBT analysis (García-Marqués et al., 2016) to detect changes in functional categories due to coordinated protein behavior. The analysis detected significantly increased pathways (positive values of Zq indicate increased protein changes) compared with the theoretical N(0,1) distribution (black curve); for comparison, the distribution of Zq values of all the proteome is also shown (red curve). The panel displays the cumulative distributions of Zq from proteins belonging to a set of representative altered categories, showing the high coordination of protein responses in these pathways.

Fig 5: Nestin+ cells regulate inflammatory cell traffic in atherosclerosis.(a) Flow cytometry plots showing the cell sorting strategy. (b) QPCR analysis of Icam1, Vcam1 and Mcp1 mRNA in BM CD45- Ter119- CD31+ endothelial cells (BMEC) and BM CD45- Ter119- CD31- stromal cells (BMSC) from ApoE-/- mice fed with chow or HFD for 2 months (n=3–7). (c) CD31 expression in BM GFP+ stromal cells from Nes-gfp;ApoE-/- mice fed with HFD for 2–3 months (n=4). (d) Experimental design of peripheral blood analysis from mice treated with tamoxifen and fed with HFD for 2 months. (e,f) Flow cytometry strategy used to identify inflammatory neutrophils, inflammatory monocytes and non-classical monocytes. (g,h) Number of non-classical monocytes, inflammatory monocytes and inflammatory neutrophils in the peripheral blood of tamoxifen-treated (g) Nes-CreERT2;Mcp1f/f;ApoE-/- mice, (h) Cdh5-CreERT2;Mcp1f/f;ApoE-/- mice and control littermates (n=3–12). Data are means±s.e.m.; n and P values are indicated; *P<0.05, unpaired two-tailed t test.