Fig. 3: Regulation of MI-HCSs by vitamin A metabolites.
a, GO term enrichment of upregulated DEGs (log2FC threshold 0.8, adjusted P value <0.05) in human control HSCs in comparison with MI HSCs based on scRNA-seq. b, Experimental design to characterize human BM HSCs isolated from healthy donors. HSCs were treated in vitro with RA metabolites (at-RA or 4-oxo-RA) or DMSO as control. c, GSEA of published human HSPC signatures in DEGs between 4-oxo-RA and at-RA treatments versus control from healthy human donors based on population RNA-seq. d, Second plating of CFU of human HSCs after in vitro cultivation with RA metabolites or control treatment. Ordinary one-way ANOVA. n = 6 Ctrl; n = 4 at-RA; n = 5 4-oxo-RA. e, scHSC division assay of HSCs isolated from BM healthy donors and in vitro cultured with RA metabolites or DMSO. Depicted P values correspond to the percentage of non-divided cells. Statistics denote comparisons between at-RA or 4-oxo-RA condition and the control condition. Ordinary two-way ANOVA. n = 7 Ctrl; n = 9 at-RA; n = 9 4-oxo-RA. f, Experimental design to characterize HSC response after at-RA in vivo treatment after MI. n = 4 vehicle; n = 3 at-RA; n = 2 sham. g, GSEA of published mouse HSPC signatures in vehicle versus sham HSCs in day-2 post-MI population RNA-seq. h, GSEA profile of HSC signature in at-RA versus vehicle HSCs upon MI. RES, running enrichment score. i, Flow-cytometry-based analysis of HSC cell cycle of sham, MI + vehicle and MI + at-RA conditions. The percentage of cell-cycle phases (G0, G1 and G2/S/M) is shown. Depicted P values correspond to the percentage of cells in the G0 phase. Statistics denote comparisons between vehicle or at-RA condition and the sham condition. Ordinary two-way ANOVA. n = 6 sham; n = 11 vehicle; n = 13 at-RA. j, scHSC division assay after 48 h in sham, MI + vehicle and MI + at-RA HSCs. The percentage of cells is shown. Depicted P values correspond to the percentage of non-divided cells. Statistics denote comparisons between vehicle or at-RA condition and the sham condition. Ordinary two-way ANOVA. n = 6 sham; n = 11 vehicle; n = 13 at-RA. k,l, Flow-cytometry-based analysis of leukocyte frequencies during EH in the acute phase at day 2 after MI. The percentage of leukocyte cell frequencies is depicted in BM and myocardium. Ordinary one-way ANOVA (k: n = 12 sham; n = 11 vehicle; n = 12 at-RA; l: n = 9 sham; n = 11 vehicle; n = 12 at-RA). GMPs, granulocyte-macrophage progenitors. m, Quantification of myocardium CD11bpos immunohistochemistry (IHC) stainings of vehicle and at-RA condition in the chronic phase at day 28 after MI. Two-tailed unpaired t-test. n = 11 vehicle; n = 8 at-RA. n, The percentage of EF of LV based on echocardiography performed during the acute phase at day 1 after MI and during the chronic phase at day 21 after MI for each condition. Ordinary one-way ANOVA. n = 5 sham; n = 9 vehicle; n = 9 at-RA. LV, left ventricle. o, Expression profiles of RA receptors in BM and cardiac cells. Left: real time (RT)-qPCR in BM HSPCs, differentiated immune cells and niche cells. Normalized mean relative to Oaz1 expression and relative to HSCs is shown. n = 3 per condition. Ct, cycle treshold. Right: heatmap of normalized counts in cardiac cell population RNA-seq dataset. n = 3 Mono; n = 4 Mφ; n = 3 FB; n = 4 EC. Mono, monocytes; Mφ, macrophages; FB, fibroblasts; EC, endothelial cells. p, GSEA of published mouse inflammatory-macrophage signature in cardiac monocytes and macrophages isolated from mice after 24 h in vivo treatment with at-RA or DMSO (vehicle), based on population RNA-seq. n = 3 Mono vehicle; n = 4 Mono at-RA; n = 4 Mφ vehicle; n = 3 Mφ at-RA. In g–l, cells were isolated in the acute phase at days 2–3 after MI. Data are presented as mean ± standard deviation. In c–e, n indicates the number of human BM donors per condition (biological replicates). In g–p, n indicates the number of biological replicates per condition. For c–e and j–p, two or more independent experiments were performed.
