Fig. 4. DNMT3A^Mut cells exposed to FBM maintain an HSC pool characterized by an inflammatory phenotype.

a Cells from DNMT3A^Mut and DNMT3A^WT were injected to mice (n = 3) with fatty bone marrow (FBM) and normal bone marrow (NBM). Three days after injection lin- KIT+ (LK) cells were isolated from mice bone marrow (BM) and underwent single-cell RNA-Seq analysis. MetaCell algorithm was used to assign different single cells to Metacells with unique gene programs and cell types⁴⁴. Gold, hematopoietic stem cells (HSCs); dark green, common myeloid progenitors (CMP); light blue, common lymphoid progenitors, (CLP); cyan, dendritic progenitors (DcP); gray, multipotent progenitors (MPP); dark olivegreen, monocyte progenitors (MonoP); pink, megakaryocyte progenitors (MegK); red, erythroid progenitors (Ery); grey4 (Unknown). Conditions: normal bone marrow (NBM); wild-type (wt); fatty bone marrow (FBM); naive cells—are cells extracted directly from BM of respective mice without transplantation. cre is the cre control. b While all injected cells show a marked reduction in HSCs after transplantation DNMT3A^Mut cells exposed to FBM maintain their HSC pool which is significantly higher than all other condition (which were transplanted). Exact fisher test was used ****P < 0.0005 to compare proportions of HSCs between the groups. FBM_MUT vs. FBM_WT P = 2.3 e10⁻⁹. c Ranked GSEA analysis on differentially expressed genes between DNMT3A^Mut cells exposed to FBM cluster and other clusters exposed significant enrichment of inflammatory pathways one of them was the IL-6, JAK and STAT3 response gene set. An expression score for every single cell was calculated based on the expression of each of the genes in the IL-6 gene set. All comparisons were performed using a two-tailed, nonpaired, nonparametric Wilcoxon rank-sum test with 95% confidence interval with FDR multiple hypothesis. *P < 0.05, ****P < 0.0005. FBM_mut vs. FBM_wt P = 0.01281, FBM_mut vs. Naive_mut_1y P = 2.18E-09. d Multiplex cytokines assay (FirePlex-96 Key Cytokines (Mouse) Immunoassay Panel (ab235656)) of 17 common cytokines analyzed by FACS-based multiplex method of serum from NBM, FBM and following PPARγi administration to NSG mice, without any cell’s transplantation. Data were analyzed by two-way ANOVA test—Sidaks multiple comparison test. The figure displays the P values. NBM n = 4, 3, 3, 4, 4, 4, 4, 3, 4, 4, 3, 4, 6, 4, 5, 4, 6. FBM n = 9, 7, 6, 9, 6, 6, 7, 7, 8, 8, 7, 9, 9, 6, 7, 9, 8. FBM+PPARγi n = 5, 3, 3, 5, 3, 3, 3, 3, 3, 3, 3, 3, 5, 5, 5, 5, 3. e Multiplex cytokines assay (FirePlex-96 Key Cytokines (Mouse) Immunoassay Panel (ab235656)) of 17 common cytokines analyzed by FACS-based multiplex method of BM from NBM, FBM and following PPARγi administration to NSG mice, without any cell’s transplantation. Data were analyzed by two-way ANOVA test—Sidaks multiple comparison test. The figure displays the P values. NBM n = 3, 4, 4, 4, 4, 4, 4, 4, 4, 4, 3, 4, 3, 4, 4, 4, 4. FBM n = 5, 5, 5, 5, 5, 5, 5, 3, 5, 5, 3, 4, 4, 4, 5, 5, 4. FBM+PPARγi n = 4 for all. f, g FACS-based multiplex method of NBM, FBM, and following PPARγi administration to NSG mice transplanted with 1-year-old DNMT3A^Mut or DNMT3A^WT BM-derived cells. Each bar represents 4 to 5 mice. Data were analyzed by two-way ANOVA test—Sidaks multiple comparison test. The figure displays the P values. f NBM n = 4 for all. FBM n = 4, 4, 4, 4, 4, 4, 4, 4, 4, 5, 4, 5, 5, 5, 5, 5, 4. FBM+PPARγi n = 4, 3, 3, 4, 3, 3, 3, 3, 3, 3, 3, 3, 3, 4, 4, 4, 3. g NBM n = 5 for all, except for IL-6 n = 4. FBM n = 5 for all. FBM+PPARγi n = 5, 3, 3, 5, 4, 3, 5, 5, 5, 5, 5, 5, 4, 3, 3, 5, 5. Source data are provided as a Source Data file.