Single-Cell Proteomics Reveals Specific Cellular Subtypes in Cardiomyocytes Derived From Human iPSCs and Adult Hearts

Figures

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  Fig. 1. Single-cell proteome trajectory of differentiating cardiomyocytes.A, workflow from iCM differentiation to single-cell proteomics acquisition. B, UMAP of the proteome of single cells at each differentiation time point. C, dot plot of cardiomyocyte marker proteins expressed at each time point; UMAPs of LN28A (stem cell marker) and MHY6 (cardiomyocyte marker). D, Pseudotime trajectory of iCM differentiation and expression of LN28A and MHY6 at the single-cell level. E, UMAP of only Day-0 iPSCs identified in 3 Leiden clusters; UMAPs colored by markers of cell cycle stage, LN28A, and Tra-1-60; Dot plot represents the top seven proteins differentially expressed in subcluster 1 of iPSCs; Immunocytochemistry analysis of Lin28a expression in iPSC colonies at Day0. F, UMAPs of only Day-21 iCMs identified in 3 Leiden clusters; Dot plot top 5 proteins expressed in each cluster. G, Volcano plots of Day-21 iCMs cluster 1 vs cluster 0, cluster 1 vs cluster 2, and cluster 0 vs cluster 2. (enriched proteins are highlighted in red; depleted proteins are in blue). The color bar labels in all UMAP figures represent log fold change values of protein intensity.

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  Fig. 2. Single-cell proteomics of aCMs.A, workflow of aCM isolation to single cell proteomics. B, representative images of aCMs in the dispensing capillary before sorting. C, Cell size comparison between aCM and iCM (aCMs=161 cells; iCMs=187 cells). D, proteome overlaps between aCMs and Day21 iCMs. E–G, radar plots comparing ClueGO enrichments in cellular components (E), biological processes (F), and pathways, in aCMs unique proteins (green), iCM unique proteins (orange), and the common aCM and iCM overlapping proteins (blue). H–J. UMAPs of individual aCM proteomes colored by the location isolated (H), two Leiden clusters (I), and protein count (J), in human1. K, cell size comparison between Leiden clusters 0 and 1 human1 aCMs. L-M. UMAPs of individual aCM proteomes colored by the location isolated (L), two Leiden clusters (M), and protein count (N), in human2. O, cell size comparison between Leiden clusters 0 and 1 human2 aCMs; p > 0.05, not significant by t test. P–R. ClueGO protein enrichments of cluster0 aCMs in cellular components (P), biological processes (Q), and pathways (R), compared to cluster1 aCMs in human1 (blue) and human2 (orange), respectively.