A human-on-human assay for detecting anti-myocardial antibodies in patients with myocardial disease

Figures

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  Figure 1. Schematic representation of the human-iPSC-CM-derived immunofluorescence assay workflow. Induced pluripotent stem cells of healthy donors are differentiated into cardiomyocytes. Patient’s or healthy control samples are added to the cells together with secondary antibodies for immunofluorescence staining. Fluorescence microscopy is performed, and the acquired images are quantitatively analyzed. Created in Biorender.com.

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  Figure 2. Dose dependent binding of Patients serum to human derived cardiomyocytes at day 90 after start of differentiation. (A) Serum of healthy control binding to cardiomyocytes with 1:10, 1:50, and 1:100 dilution. (B) Serum of patient after myocardial infarction with 1:10, 1:50 and 1:100 dilution. (C) Serum of Myocarditis patient with 1:10, 1:50, and 1:100 dilution. (A-C) Serum (anti-human IgG A555) in green, alpha-actinin (anti-mouse IgG A488) in magenta, and Hoechst staining for nucleus in blue. Scale 10 µm. (D) Normalized intensity for serum (green) and nucleus (blue) for healthy control (ctrl) derived from (A), MI derived from (B), and Myo derived from (C) (mean intensity per cell). Data is represented as bar graph with mean +/- SD. *p < 0.05 significant differences by one-way Anova with Tukey post-hoc test. Pictograms from BioRender.com.

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  Figure 3. Translation to low expert labs with widefield fluorescence microscopy. (A) Widefield fluorescence image of 1:10 diluted serum of myocardial infarction patient. B) Widefield fluorescence image of 1:10 diluted serum of myocarditis patient. C) Widefield fluorescence image of 1:10 diluted serum of healthy donor. (A-C) Serum (anti-human IgG A555) in green, alpha-actinin (anti-mouse IgG A488) in magenta, and Hoechst staining for nucleus in blue. Scale: 20 µm. (D) Normalized intensity for serum (green) and nucleus (blue) for MI derived from (A), Myo derived from (B), and healthy control (ctrl) derived from (C) (mean intensity per cell). Data is represented as bar graph with mean +/- SD. *p < 0.05 significant differences by one-way Anova with Tukey post-hoc test. (E, F) Normalized intensity for serum (green) and nucleus (blue) for MI and healthy control (ctrl) from two independent experiments. Pictograms from BioRender.com.

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  Figure 4. Comparison of five patients per group. Normalized intensity for serum (A) and nucleus (B) of 10 cells from 5 different patients per group (MI) in comparison to the healthy control (Ctrl). Mean of each patient and mean of each healthy control represented as black square. Data is represented as bar graph with mean +/- SD. *p < 0.05 significant differences by one-way Anova with Tukey post-hoc test.