Cardiomyocyte Transplantation after Myocardial Infarction Alters the Immune Response in the Heart

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  Figure 1. Gating strategy for identifying the specific immune subpopulations using Flow cytometry. Firstly, the mononuclear cells (MNC) that express CD45 were gated and only single cells were included into the analysis. Dead cells were excluded using Zombie-AmCyan dye. The remaining viable CD45+ MNCs were further characterized based on the expression of specific markers using two different panels: (A) Myeloid panel, (B) T cell panel. In both cases, only CD45 expressing mononuclear single-cell suspensions was included. In the myeloid panel, the live cells were then grouped based on their specific marker expression: R1: CD11b+ myeloid cells, R2: Dendritic cells, R3: NK cells, R4: Monocytic cells, R5: Neutrophils, R6: Ly6Chi or commonly known as M1 cells, R7: Ly6Clo or commonly known as M2 cells, R8: Fetal liver-HSC derived resident macrophages, R9: Monocyte derived macrophages, R10: Monocytes, R11: Yolk-sac derived resident macrophages. In the T cell panel, the viable CD45+ cells were then gated for T cells based on the expression of CD3e. The T cells were then further grouped based on their specific marker expression combinations: R1: CD4+ helper T cells, R2: CD4+ CD8+ T cells, R3: CD8+…

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  Figure 2. Cardiomyocyte transplantation alters the immune response in the heart after myocardial infarction (MI). (A) Experimental setup. (B–I) Flow cytometric analysis of the various immune cell populations in the heart (B–H) and spleen (I) of C57BL/6J mice four and seven days following MI and cardiomyocyte transplantation (MIC). The various cell populations were identified based on the strategy presented in Figure 1. n = 7. Values are represented as the mean ± SEM. Significance was calculated using the Mann–Whitney test. * p < 0.05, ** p < 0.01.

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  Figure 3. Syngeneic cardiomyocyte transplantation after MI leads to improved cardiac pump function and macrophage infiltration with no improvement in fibrosis and capillary density. Assessment of (A) Left Ventricular Ejection Fraction (LVEF,%), (B) End Systolic Volume (ESV, µL), (C) End Diastolic Volume (EDV, µL) using MRI (n = 7) and pressure characteristics, (D) dP/dT max (mmHg/s) using conductance catheter (n = 6–7) four weeks after MI. (E) A tile scan of the heart four weeks after cell transplantation with an arrow pointing towards GFP signals observed at the injection site. Scale bar represents 200 µm. (F) Assessment of fibrotic area in the heart after four weeks using Fast Green/Sirius Red staining (n = 7). The heart slices were observed using a 10× objective. (G) Assessment of CD31+ cells in the remote area of the heart four weeks after MI (n = 7). CD31+ cells were stained red and the nuclei were stained blue using DAPI. Scale bar represents 20 µm. (H) Assessment of CD68+ cells in the remote area of the heart four weeks after MI (n = 7). CD68+ cells were stained red and the nuclei were stained blue using DAPI. Scale bar represents 20 µm. Values are represented as the mean ± SEM.…

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  Figure 4. Transcriptomic analysis of the heart and blood. C57BL/6J mice were subjected to MI, MIC and seven days later, the RNA from the heart and blood was isolated and analyzed via a ClariomTM D microarray. (A) The log(2) scale expression signals of the differentially expressed (DE) transcripts between the cell treated (BL6MIC) and infarct (BL6MI) groups are illustrated as Manhattan plots. DE transcripts in the blood are shown in red while the DE transcripts from the heart are shown in blue. Transcripts are considered DE, if they show a two-fold change and have a p-value < 0.05. (B) The relative contribution of the various categories of RNA to these differentially expressed transcripts in the heart and blood (n = 3).

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  Figure 5. Cardiomyocyte transplantation has varied effects on the gene expression in the heart and blood. Heatmap of annotated differentially expressed (DE) transcripts found between BL6MIC and BL6MI in the heart (A) and blood (B). Transcripts are considered DE if they show a two-fold change and have a p-value < 0.05. Upregulated targets are colored red while the downregulated targets are colored green.

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  Figure 6. Gene Ontology (GO) enrichment and network analysis reveals the pathways influenced by cardiomyocyte transplantation. GO enrichment analysis expressed as % terms per group was performed on differentially expressed (DE) targets in the heart (A) and blood (B). (C) Network analysis using the GO terms of these targets. In the network, the transcripts from the heart and the corresponding GO terms are represented in blue circles with the transcripts from the blood and their corresponding GO terms being represented in red circles. (D) The respective GO ID and GO terms of these DE targets in the heart and blood.