Molecular Mechanisms of Exosomes From Human iPSC‐Cardiomyocytes and Mesenchymal Stem Cells in Restoring the Injured Myocardium

Figures

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  Figure 1. Cardiac MRI of porcine hearts treated with PBS (n=4), MSC‐Exo (n=5) or iCM‐Exo (n=6). A, Left ventricular ejection fraction, (B) left ventricular end diastolic volume, (C) left ventricular end systolic volume, and (D) infarct size as an area percentage of delayed gadolinium enhancement MRI‐positive signal with a representative of infarct size. Statistical significance was determined by Kruskal–Wallis (iCM‐Exo, n=6; MSC‐Exo, n=5; PBS n=4) with multiple comparisons. Median with interquartile range is shown. Only significance between control and treatment groups at weeks 2 and 4 are shown (*P<0.05, **P<0.01). iCM‐Exo indicates exosomes derived from induced pluripotent stem cell–derived cardiomyocytes; LVEDV, left ventricular end‐diastolic volume; LVEF, left ventricular ejection fraction; LVESV, left ventricular end‐systolic volume; MRI, magnetic resonance imaging; and MSC‐Exo, exosomes derived from mesenchymal stem cells.

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  Figure 2. Total myocardial and peri‐infarct viability measured by maganese uptake of porcine hearts treated with PBS (n=4), MSC‐Exo (n=5), or iCM‐Exo (n=6). A, Manganese‐enhanced MRI percentage viability and (B) peri‐infarct region percentage viability. Statistical significance was determined by Kruskal–Wallis (iCM‐Exo, n=5; MSC‐Exo, n=4; PBS n=3) with multiple comparisons. Median with interquartile range is shown. Only significance between control and treatment groups within each week are shown (*P<0.05). iCM‐Exo indicates exosomes derived from induced pluripotent stem cell‐derived cardiomyocytes; MEMRI, manganese‐enhanced MRI; MRI, magnetic resonance imaging; MSC‐Exo, exosomes derived from mesenchymal stem cells; and PIR, peri‐infarct region.

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  Figure 3. Gene expression between iCM‐Exo (n=6) and MSC‐Exo (n=5). (A) Venn diagrams of differentially expressed genes between iCM‐Exo, MSC‐Exo, and PBS treatments. These results highlight distinct transcriptional responses of iCM‐Exo and MSC‐Exo while identifying shared molecular signatures relevant to cardiac repair mechanisms. Dot plot of top enriched GO:BP enrichment analysis of DEGs associated with (B) upregulated and (C) downregulated genes in the iCM‐Exo treatment group and top enriched GO:BP terms associated with (D) upregulated and (E) downregulated genes in the MSC‐Exo treatment groups. The x axis represents fold enrichment, while the y axis lists the enriched biological processes. Dot size indicates the number of genes involved in each process, and dot color represents the statistical significance of enrichment (−log10[FDR]). FDR indicates false discovery rate; GO:BP, Gene Ontology Biological Process; iCM, induced pluripotent stem cell–derived cardiomyocyte; and MSC, mesenchymal stem cell.

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  Figure 4. Pathway analysis of iCM‐Exo (n=6) and MSC‐Exo (n=5). A, Unique and overlapping pathways between iCM‐Exo and MSC‐Exo treatments by regulation type. Bar graph depicting the number of pathways uniquely enriched or shared between iCM‐Exo and MSC‐Exo treatments, categorized as upregulated (blue) or downregulated (red). Overlapping pathways represent shared biological processes modulated by both exosome types while iCM Unique and MSC Unique pathways reflect distinct mechanisms specific to each treatment. The analysis reveals a larger number of pathways uniquely associated with iCM‐Exo treatment, particularly in upregulated pathways, highlighting its broader transcriptional impact compared with MSC‐Exo. B, Hierarchical clustering of top 10 upregulated and downregulated KEGG Pathways in iCM vs MSC treatments. This hierarchical clustering tree visualizes the top 10 KEGG pathways enriched in porcine tissue treated with iCM‐Exo vs MSC‐Exo. Upregulated pathways are indicated by red dots, and downregulated pathways by blue dots. The tree shows the relative significance and gene overlap among the pathways with smaller P values indicating more significant enrichments. KEGG pathway network…

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  Figure 5. Hierarchical clustering and heatmap of gene expression. (A) GO and biological processes are shown by the heatmap displaying hierarchical clustering of the top 2000 genes ranked by standard deviation across all samples and highlighting the transcriptional differences between iCM‐Exo, MSC‐Exo, and PBS treatments. Gene expression is centered by subtracting the average expression for each gene with colors representing upregulated (red) and downregulated (blue) genes. Hierarchical clustering was performed using Pearson's correlation coefficient (1−r) as the distance metric and average linkage as the clustering method. K‐means clustering (k=2) was applied, as determined by the elbow plot. Adjacent to the heatmap, hierarchical trees illustrate enriched GO:BPs for upregulated and downregulated genes in each cluster. (B) Heatmap of differentially expressed miRNAs between iCM‐Exo and MSC‐Exo treatments. Heatmap illustrating the expression patterns of miRNAs that were significantly differentially expressed between iCM‐Exo and MSC‐Exo groups. Rows represent individual miRNAs, and columns represent biological replicates of each exosome type. Expression levels are color‐coded, with red…

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  Figure 6. Pathway enrichment analysis. (A) iCM‐Exo upregulation of miRNAs is shown in the bar plot depicting the top 20 enriched GO:BP terms derived from the Genes Union method. The x axis represents the −log10(FDR) of each pathway, indicating the significance of enrichment, while the y axis lists the enriched terms. The number of target genes contributing to each pathway is annotated beside each bar. Heatmap displaying enriched pathways identified via the Pathways Union method. Each cell represents the significance (−log10[FDR]) of a specific miRNA‐term pair, allowing visualization of how individual miRNAs contribute to enriched pathways. The hierarchical clustering highlights shared and distinct pathway involvement among miRNAs upregulated in iCM‐Exo. (B) iCM‐Exo downregulation of miRNAs is shown by the bar plot depicting the top 20 enriched Gene Ontology Biological Process (GO:BP) terms derived from the Genes Union method. The x axis represents the −log10(FDR) of each pathway, indicating the significance of enrichment, while the y axis lists the enriched terms. The number of target genes contributing to each pathway is annotated beside each bar. Heatmap displaying enriched pathways…