Serum of patients with acute myocardial infarction prevents inflammation in iPSC-cardiomyocytes

Figure 1. (A). mRNA expression of the pluripotency gene POU5F1 and of the cardiac marker troponin I (TNNT2) during differentiation of hiPSC into cardiomyocytes. The fold change over time relative to the expression of the housekeeping gene GAPDH is presented. Mean of 3 technical replicates of 3–6 biological replicates of 3 different differentiation rounds is shown. Whiskers display standard error. (B). Percentage of marker-expressing iPSC-cardiomyocytes (% of TNNT2-positive cells) at day 25 of differentiation. Mean of n = 5–12 measurements per marker. Whiskers display standard deviation. (C). CD162, CD31, CD62, and CD102 (blue) were not found on iPSC-cardiomyocytes.

Figure 2. (A). Expression levels (median fluorescence intensity) of CD58/LFA-3, CD54/ICAM-1 and CD321/JAM-A on iPSC-cardiomyocytes (TNNT2-positive cells) at different times of differentiation. N = 5–10 measurements per marker. The mean ± standard deviation is shown. (B). Representative plots of flow cytometry for CD58/LFA-3, CD54/ICAM-1 and CD321/JAM-A (blue) showing the marker-positive sub-populations at differentiation day 25. The number of each marker subset gives the percentage of positive cells of all cells. (C). Percentage of iPSC-cardiomyocytes (% of TNNT2-positive cells) expressing CD58/LFA-3, CD54/ICAM-1 and CD321/JAM-A at different times of differentiation. N = 5–10 measurements per marker. The mean ± standard deviation is shown.

Figure 3. Immunofluorescence staining demonstrates the localization of CD58/LFA-3 (A, green), CD54/ICAM-1 (B, green) and CD321/JAM-A (C, yellow) in iPSC-cardiomyocytes (A–C: troponin T, red; nuclei, blue). Each marker is also expressed by cells other than cardiomyocytes, which can best be seen with CD58. Images were taken at 40x magnification. Scale bars represent 50 µm with the exception of CD58 (200 µm for better overview of marker expression by different cells).

Figure 4. Expression levels (median fluorescence intensity relative to cell number) on iPSC-cardiomyocytes after incubation with myocardial infarction serum (MI) or control serum (5% or 20% v/v). (A) CD58/LFA-3, (B) CD54/ICAM-1, (C) CD321/JAM-A. N = 13–26 measurements per marker. The line within box represents the median, additionally the 10th, 25th, 75th, and 90th percentile is shown; whiskers represent error bars.

Figure 5. Expression levels (median fluorescence intensity relative to cell number) on iPSC-cardiomyocytes after incubation with 5% or 20% (v/v) myocardial infarction serum with vs. without ventricular fibrillation (VF). (A) CD58/LFA-3, (B) CD54/ICAM-1, (C) CD321/JAM-A. N = 7–15 measurements per marker. The line within box represents the median, additionally the 10th, 25th, 75th, and 90th percentile is shown; whiskers represent error bars.

Figure 6. Incubation with myocardial infarction serum (MI, black) does not change electrophysiological properties (control serum – Ctr, white). RP – Resting potential, APA – action potential amplitude, Vmax – maximum depolarization velocity, APD – action potential duration. The mean of the indicated number of measurements is shown; whiskers display standard deviation.

Figure 7. Incubation with myocardial infarction serum with ventricular fibrillation (VF, black) does not change electrophysiological properties (myocardial infarction serum without ventricular fibrillation – MI, white). RP – Resting potential, APA – action potential amplitude, Vmax – maximum depolarization velocity, APD – action potential duration. The mean of the indicated number of measurements is shown; whiskers display standard deviation.