Fig. 2: HSCs contribute to inflammatory myeloid cell infiltration in the heart.

a, Experimental design for tracing the lineage of HSC responses following LAD artery ligation upon MI using the Fgd5^CreERT2 mouse model. Fgd5^CreERT2 mice express a tamoxifen-inducible CreERT2 recombinase and green fluorescent protein (ZsGreen) in the Fgd5 locus, which is highly active in HSCs. The expression of red fluorescent protein (tdTomato) is controlled by a loxP-flanked STOP cassette. Upon Cre-mediated recombination, dTomato fluorescence is observed. The dTomato expression is compared between (1) baseline mice undergoing induction without any surgical intervention, (2) non-ischaemic sham surgery mice and (3) LAD-ligated mice to simulate MI conditions. EH in Fgd5^CreERT2 mice is evaluated during the acute phase at day 3 after MI. b, Flow-cytometry-based analysis of lineage-traced BM progenitors in Fgd5^CreERT2 mice. The percentage of dTomato^pos cells is normalized to the dTomato label within the HSC compartment across the progenitors: HSCs, MPPs and MyP at baseline, after sham surgery and after MI. Ordinary two-way analysis of variance (ANOVA). Red P value, baseline versus sham; grey P value, sham versus MI; black P value, MI versus baseline. n = 6 vehicle; n = 6 sham; n = 3 baseline. c, Flow-cytometry-based plots, illustrating the percentage of lineage-traced dTomato^pos cell frequencies of myeloid cells upon MI or sham surgery in BM of Fgd5^CreERT2 mice. Ordinary two-way ANOVA. n = 6 vehicle; n = 6 sham; n = 3 baseline. Leuko, leukocytes. d, Sections of myocardium stained with DAPI after MI. The counts of dTomato^pos cells are normalized to DAPI counts. Scale bar, 100 µm. Two-tailed unpaired t-test. n = 7 sham; n = 5 MI. e, Alluvial plot of contribution from dTomato^pos and dTomato^neg, CD11b^pos cells to the myocardium upon MI. The y axis is reduced for visualization purposes. f, Flow-cytometry-based representative UMAP plots calculated on downsampled cells isolated from myocardium. The plots are representative for four sham mice and two MI mice. g, Flow-cytometry-based plots, illustrating the percentage of lineage-traced dTomato^pos leukocyte frequencies upon MI or sham surgery of Fgd5^CreERT2 mice in the myocardium. Ordinary two-way ANOVA. n = 4 sham; n = 6 MI. Leuko, leukocytes; Ly6c2hi mono, Ly6c2-high monocytes. h, Volcano plot of DEGs between dTomato^pos and dTomato^neg mouse CD11b^pos cardiac MI cells in scRNA-seq. i, UMAP plot of scRNA-seq on mouse cardiac CD11b^pos cells in MI coloured by cell type annotation. Pro-Inflam Mo/Neu, pro-inflammatory monocytes and neutrophils; reparatory Mφ, reparatory macrophages; resident Mφ, resident macrophages; APC, antigen-presenting cells; Mono, monocytes. n = 2 per condition. j, UMAP density plots of dTomato^pos and dTomato^neg CD11b^pos cells in the myocardium upon MI depicting relative cell abundance. k, Stacked bar plot of quantified relative cluster abundance in dTomato^pos and dTomato^neg CD11b^pos cells in myocardium upon MI. Data are presented as mean ± standard deviation. In b–i, n indicates the number of biological replicates. For b–e and g, four independent experiments were performed.

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