Human Cardiac Organoids for Modeling Genetic Cardiomyopathy

Figures

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  Figure 1. Illustration of hiPSC differentiation with Chemically-Defined Medium 3 (CDM3) protocol adapted from Burridge et al. [21].

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  Figure 2. Characterization of cardiomyocytes derived from WTSIi020-A and UKKi025-A. (A,B) Flow cytometry analysis of cells positive for TNNT2 and OCT4 at the beginning (day 0) and the end (day 15) of differentiation in CDM3. (C,D) Median fluorescence intensities of TNNT2+ and OCT4+ populations at day 0 and 15 of the CDM3 protocol. n = 3 biological replicates. (E,F) Heatmap plots representing RT-qPCR for markers of pluripotency (NANOG, OCT4, SOX2, KLF4, MYC), cardiac progenitors (NKX2.5) and cardiomyocytes (TNNT2, MHY6, MHY7) on day 0 and day 15. n = 3 independent biological replicates performed in technical triplicates. (G) Immunofluorescent staining images of cardiomyocytes derived from WTSIi020-A and UKKi025-A. WTSIi020-A CMs show clear formation of immature sarcomeric-like structure while UKKi025-A CMs display a disrupted structure and rather enlarged cell morphology (marked with *). Significance level A: **** poct4 < 0.001; * ptnnt2 < 0.023; B: *** poct4 = 0.0002; **** ptnnt2 < 0.0001; C: ** poct4 = 0.0015; **** ptnnt2 < 0.0001; D: * poct4 = 0.0142; **** ptnnt2 < 0.0001; E: p ≤ 0.0001; F: p ≤ 0.0002.

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  Figure 3. Modelling Adult Human Heart Organoids. (A) Generation of contractile cardiac organoids (Cor-Oids) containing Cor.4U-CMs, HCMECs and HCF at 3:5:2 cell ratio using Terasaki plates showed the formation of compact ball-like shapes after 21 days of culture. (B) Immunofluorescence staining images of Cor-Oids after 14 days of culture, labelled with cardiac-specific troponin (TNNT2), vascular cadherin (VeCADH) and vimentin (VIM), showing organoids with a lack of defined cardiac structures (T-tubules and sarcomeres). (C) Immunofluorescence staining images of Cor-Oids after 21 days of culture, labelled with cardiac-specific troponin (TNNT2), vascular cadherin (VeCADH) and vimentin (VIM), showing CMs migration toward the organoid core, promoting the formation of cardiac filament-like structures. Scale bars = 200 µm.

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  Figure 4. Determining the structural development of cardiac organoids. (A–C) Immunofluorescence images of Cor-Oids, WTS-Oids and UKK-Oids after 21 days of culture, labelled with cardiac-specific troponin (TNNT2), vascular cadherin (VeCADH) and vimentin (VIM). We identified similar structural patterns (A–C, iii–iv) in all organoid types, in terms of HCMEC (magenta) and HCF (green) distribution. Different structural development patterns were observed regarding the migration of hiPSC-CMs and the formation of cardiac filament-like structures (red), which were well-defined in Cor-Oids (A, i–ii), mostly perimetric in WTS-Oids (B, i–ii), and still in formation in UKK-Oids (C, i–ii). Scale bars = 200 µm.

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  Figure 5. Patient-specific (patho-)physiological features mirrored by cardiac organoids. (A–C) Analysis of the beating patterns and rates of Cor-Oids, WTS-Oids and UKK-Oids on day 21 (A) Cor-Oids showed a regular beating pattern with a beating rate of 82 ± 2 bpm (B) WTS-Oids showed a similar regular beating pattern, but a slower beating rate than Cor-Oids (C) UKK-Oids presented an arrhythmic beating pattern and a significant variability in their beating rate. RFI = Relative Fluorescence Intensity.