Fig. 5: Characterization of HSPCs and their effect on BM inflammation.

A Spider plot of the relative abundances of HSPC subtypes across Control (green), CHIP (purple), and MDS (orange) donors. Proportions are probit-transformed; black outlines indicate significant differences compared to Controls. Statistical test: Fisher’s exact test. B UMAP of HSPC cells showing the pseudotime and schematic outline of main differentiation trajectories. C Smoothed expression of pro-inflammatory cytokine genes CXCL8 and IL1B along the pseudotime in 3 main differentiation trajectories of HSPCs. Smoothing and 95% confidence intervals are calculated by the function geom_smooth from ggplot2 (posterior distribution of coefficients of a cubic spline generalized additive regression model used for smoothing). D UMAP of HSPCs showing the SpliceUp-predicted SF3B1 mutational status (SF3B1^WT blue, SF3B1^MUT red). Dashed box highlights the erythroid progenitors, further analyzed in (E, F). E Differential expression between SpliceUp-predicted SF3B1^WT and SF3B1^MUT erythroid progenitors. Statistical test: DESeq2. Red dots: adjusted p value < 0.05. F Gene Set Enrichment Analysis (GSEA) results with the Biological Process ontology from Gene Ontology database showing the top 20 significant sets enriched between SF3B1^WT and SF3B1^MUT erythroid progenitors. G Schematic visualization of the primary HSPC-MSC co-culture experiment. BM MSCs were cultured either alone or with CD34⁺ HSPC from Control, CHIP or MDS donors (ratio 1:1). After 96 hrs, the supernatant was collected and subjected to subsequent protein quantification using an Olink panel. The cells were harvested and subjected to scRNA-seq. Created in BioRender. Prummel, K. (https://BioRender.com/v0w0jhd). H Olink quantification of 4 secreted cytokines after 96 hrs of co-culture. Mean normalized protein expression (NPX) values (mean of 3 experimental replicates) across conditions (MSC mono-culture (MSC-only), Control CHIP, and MDS, n = 3/group) are displayed. Medians with 95% confidence intervals are shown. Statistical test: one-way ANOVA, Tukey’s test. I scRNA-seq of co-cultures showing normalized RNA expression of cytokines in (H) across the stromal (triangles) and HSPC (circles) compartments after MSC mono-culture (MSC-only), and Control and MDS co-cultures (n = 3/group). Medians with 95% confidence intervals are shown. Statistical test: two-sided unpaired Student’s t-test. A, E, F P values were adjusted using Benjamini–Hochberg procedure. Source data are provided in the Source Data file and Supplementary Data 7. n.s. not significant.