18F-FDG PET-Based Imaging of Myocardial Inflammation Predicts a Functional Outcome Following Transplantation of mESC-Derived Cardiac Induced Cells in a Mouse Model of Myocardial Infarction

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  Figure 1. Gating strategy for identifying the different immune populations in the heart. Mononuclear cells expressing CD45 were gated and doublets (FSC-W vs. FSC-A) were excluded. Dead cells were excluded by Zombie aqua. The live single CD45+ cells were then grouped into R1, CD11b+ myeloid cells (CD45+/CD11b+/CD11c−); R2, dendritic cells (CD45+/CD11b+/CD11c+); and R3, NK cells (CD45+/CD11b−/CD11c−/NK1.1+) based on their relative expression of CD11b and CD11c. R5, neutrophils (CD45+/CD11b+/CD11c-/Ly6Ghi) were then excluded from R1 based on their Ly6G expression. The remaining R4 monocytic cells were then further characterized into R6, Ly6Chi or commonly known as M1 cells (CD45+/CD11b+/CD11c−/Ly6Glo/Ly6Chi); R7, Ly6Clo or commonly known as M2 cells (CD45+/CD11b+/CD11c−/Ly6Glo/Ly6Clo) based on their Ly6C expression; and into R8, fetal liver HSC-derived resident macrophages (CD45+/CD11b+/CD11c-/Ly6Glo/CCR2−/MHC-IIhi); R9, monocyte derived macrophages (CD45+/CD11b+/CD11c-/Ly6Glo/CCR2+/MHC-IIhi); R10, monocytes (CD45+/CD11b+/CD11c−/Ly6Glo/CCR2+/MHC-IIlo); and R11, yolk sac-derived resident macrophages (CD45+/CD11b+/CD11c−/Ly6Glo/CCR2−/MHC-IIlo) based on their CCR2 and MHC-II expression. These…

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  Figure 2. Representative images visualizing our volumes of interest (VOI) positioning strategy: (A) Myocardium of healthy mice anaesthetized with isofluorane can be clearly delineated and served as a reference for VOI positioning. (B and C) Anaesthesia with ketamine/xylazine. A VOI of 5 µL was positioned in both anterolateral wall (infarct area, green) and remote area (inferobasal, blue). Thereby both anatomical landmarks form the CT scan and the image of healthy myocardium (A) was used.

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  Figure 3. Characterization of cardiac-induced cells: (A) Analysis of mRNA expression after six days of cardiac differentiation shows a decrease of the pluripotency marker Oct4, whereas cardiac markers are upregulated. The mRNA expression was normalized to the expression of the house keeping gene hprt. Values are expressed as fold increase as compared with day 0. Values are presented as mean ± SEM. (B) Starting at day 7 of the cardiac differentiation protocol, beating foci per embryoid bodies (EB) were counted until the end of differentiation (n = 3). Values are presented as mean ± SD. p-value was calculated using the student t-test.

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  Figure 4. Representative images of healthy mice anaesthetized with isofluorane (upper row) and ketamine/xylazine (lower row). Uptake of 18F-FDG is effectively suppressed by the use of ketamine/xylazine as can be seen in axial, coronal, and sagittal planes, as well as the polar maps.

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  Figure 5. Sample axial, coronal, and sagittal myocardial 18F-FDG images at five days post-surgical myocardial infarction (MI) induction. Isofluorane leads to high tracer accumulation in the healthy myocardium, whereas the infarcted area can be clearly identified as an area of low glucose metabolism (upper row). When using ketamine/xylazine, the most intense tracer accumulation is detected in the area of the border zone, whereas 18F-FDG uptake in healthy myocardium remains suppressed.

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  Figure 6. Regional quantitative analysis of 18F-FDG uptake five days after myocardial infarction in the whole heart, the infarct and the remote myocardium. Mice were anaesthetized with isofluorane or ketamine/xylazine, respectively. Values are presented as mean ± SD. p-value was calculated using the student t-test.

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  Figure 7. Sample axial, coronal, and sagittal myocardial 18F-FDG images at five days post-surgical MI induction and MI induction plus cell transplantation (MIC). Mice were anaesthetized with ketamine/xylazine. The MI group showed intense tracer accumulation in the border zone, whereas in cell-treated animals the highest tracer accumulation was found in the remote area.

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  Figure 8. Representative 17-segment tomographic polar maps visualizing distinctive differences of FDG-distribution in the left ventricle between the groups. The apex is in the middle and the anterior wall at top, the inferior wall at bottom, the septum in left and the lateral wall in the right. High tracer uptake is visualized by the yellow colors, lower upake by red and black. (A) MI group anaesthetized with isofluorane, the infarct region shows low FDG uptake as compared with remote myocardium; (B) MI group anaesthetized with ketamine/xylazine, the most intense FDG uptake came in the border zone; and (C) MI + cardiac induced cells (CiC) group anaesthetized with ketamine/xylazine shows a change of the FDG uptake to a pattern, which is similar to (A) with low FDG accumulation within the infarct region and highest uptake in the remote area.

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  Figure 9. Regional quantitative analysis of 18F-FDG uptake five days after myocardial infarction in the whole heart, infarct, and remote myocardium. Both MI and MIC group were anaesthetized using ketamine/xylazine. Values are presented as mean ± SD. p-value was calculated using the student t-test.

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  Figure 10. Improved cardiac function after transplantation of embryonic stem cells (ESC) derived CiC following acute myocardial infarction at 3 weeks following MI. (A) Magnetic resonance imaging analyses of infarcted animals receiving either CiC (MIC) or matrigel and healthy animals and (B) left ventricular ejection fraction was significantly higher in CiC animals vs. control animals (n = 6, p-value was calculated using the student t-test).

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  Figure 11. Effect of CiC transplantation on the percentage of various immune cell subpopulations in the heart based on flow cytometric analysis. Mice were subjected to MI/MI+ cells and the remote and infarct area of the hearts were dissected, digested, and the isolated mononuclear cells were stained using various antibodies. These immune cell populations were then characterized using the gating strategy described before and represented as three major groups. (A) CD11b+ and NK cells, (B) populations based on their CCR2/MHC-II expression, and (C) contribution of the populations towards the M1 (Ly6Chi) and M2 (Ly6Clo) cells, n = 5. Values are represented as mean ± SEM. Significance was calculated using the Mann–Whitney test.