Extended Data Fig. 7: hESC-macrophages reduce accumulation of mitochondrial proteins in cardiac microtissues.
Cytometry by time-of-flight (CyTOF) was performed on microtissues (HT-Biowires) with or without hESC-macrophages on day 3 post-seeding. n = 6 replicates per group, each replicate represented 8 pooled microtissues, experiment performed once. (a) UMAP visualization of 580,776 live and dead cells following standard quality control filtering, split by replicate in each group. (b) Percentage of Dead ATP5hi group relative to all events in each replicate. (c) Number of cardiomyocytes or fibroblasts acquired in each replicate in microtissues with or without hESC-macrophages. (d) Percentage of ATP5Amid cells relative to the number of cardiomyocytes (left) or the number of cardiomyocytes and fibroblasts (right) in microtissues with or without hESC-macrophages. (e) Pathways downregulated in microtissues with hESC-macrophages on day 14 from mass spectrometry-based proteomics data (as in Fig. 3). (f) Normalized expression of DNA 1 or DNA 2 in each cardiomyocyte subcluster (averaged in each replicate) in microtissues with or without hESC-macrophages. (g) Normalized expression live-dead label in each replicate (averaged) in CM-4 from microtissues with or without hESC-macrophages. (h) Microtissues were seeded with hESC-cardiomyocytes and fibroblasts with or without hESC-macrophages. On day 3 post-seeding, microtissues were labelled with the MitoTracker dye and flow cytometry was performed. Geometric mean fluorescence intensity (MFI) of MitoTracker in total cells, cardiomyocytes (CD45−CD14−), or fibroblasts (CD45dimCD14dim) is shown. n = 6 microtissues per group, two experiments shown (MitoTracker Green and MitoTracker Deep Red). (i) Normalized expression of cardiac troponin T (cTnT) in each replicate (averaged) for all cardiomyocyte subclusters (CyTOF data) in microtissues with or without hESC-macrophages. CM: cardiomyocyte; FB: fibroblast; MF: macrophage. Two-way ANOVA with P values adjusted for multiple comparisons using the Šídák method (B, F, I), or unpaired two-tailed t-test (C, D, E, G, H). *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent mean ± s.e.m.
