Description of the mold and seeding procedure. A. Micromachined stainless steel mold used to shape the gel - Design (left) and scanning electron microscopy image (right) of the mold – Scale bar: 500 μm. B. Molded PEG gel (left - scale bar 2 mm) and zoomed view of a pillar from its PDMS replica (middle-scale bar 100 μm). Design and size of the pillar (right). C. Timeline of the seeding procedure. D. Cardiac rings in a well one day after seeding. Images stitched with ImageJ plugin - Scale bar: 1 mm. E. Representative compaction of a ring with time after seeding (from day 0 to day 14), in brightfield - Scale bars: 200 μm.

Figure 1—figure supplement 1. Measurement of the Young’s Modulus of PEG gel

Figure 1—figure supplement 2. Measurement of the efficiency of differentiation the the iPSCs into cardiomyocytes.

Figure 1—video 1. Multiple rings beating at D14 - Brightfield imaging x4 magnification.

Composition and structure of the cardiac rings. A. Example brightfield images of cardiomyocytes seeded with or without fibroblasts one day after seeding - Scale bar: 200 μm. B. Number of full cardiac rings per well in time according to the hiPSC-CMs:fibroblasts ratio they contain (4:1,3:1 or 2:1). C. Confocal imaging of immunostained tissues at different heights: cardiac ring (top panel), and basal layer (bottom panel) of a tissue at 40X magnification. Vimentin, stained in green, corresponds to fibroblasts, troponin T, in red, is specific to cardiomyocytes, and DAPI is in blue. Scale bars: 100 μm. D. Picture of the immunostained contractile fibers at 63X magnification. Vimentin (green), Troponin T (red) and DAPI (blue) – Scale bar: 50μm. E. 3D reconstruction of a ring. x, y and z scale bars: 100 μm.

Figure 2—figure supplement 1. Larger scale pictures of panel C.

Figure 2—video 1.3D reconstruction of a ring

A. Principle of the in-house Matlab code used for contractility analysis: detection of the central pillar and monitoring of the evolution of its area in time, calculation and plot of the strain ϵA in time (ratio between the contraction amplitude in time and the maximum area of the central pillar) – Representative plots of the strain in time and its derivative in time, for a tissue at day 14. B. to E. Evolution of beating parameters through time after seeding at days 1, 3, 7, 10 and 14. The changes of all the parameters through time are significant (p<0.0001 - ANOVA for repeated measures -D1: n=57, D3: n=59 D7: n=47, D10: n=43, D14: n=36 tissues, from 3 differentiations). Beating parameters at each time point are compared to their value at day 1. B. Evolution of beat rate through time after seeding (***: p<0.002). C. Evolution of contraction stress through time after seeding (****: p<0.0001). D. Evolution of maximum contraction speed through time after seeding (****: p<0.0001). E. Evolution of maximum relaxation speed through time after seeding (****: p<0.0001).

Figure 3—figure supplement 1. Strain ϵA developed by the rings at D14.

Figure 3—video 1. Contractility of a ring at day 14.

Physiological and drug testing on the cardiac tissues. Effect of the concentration in extracellular calcium (A.), verapamil (B.), isoproterenol (C.) or dofetilide (D.) on tissues contractility: beat rate, contraction stress and the maximum contraction and relaxation speeds. These parameters are expressed as a ratio between their value for each concentration and the value at basal state ([Ca2+]=0.5 mM for calcium test and [Drug]=0M for drug testing). An ANOVA for repeated measures was carried out for each parameter. For each test, the value of each parameter at each concentration is compared to its value at the minimal concentration of the drug (respectively [Ca2+]=0.5mM, [Verapamil]=10-9 M, [Isoproterenol]=10-9 M and [Dofetilide]=10-9 M). E. Poincaré plot for 0 nM, 1 nM, 3 nM and 7 nM of dofetilide. For each concentration of each drug, more than 20 tissues from 3 different concentrations could be analyzed. Data is presented as mean ±SEM. *: p<0.05;**: p<0.01;***: p<0.001;****: p<0.0001.

A. Procedure to measure the Young’s Modulus of a PEG-gel disk by pipette aspiration. B. Young’s modulus of 5% PEG gels (n=44).

Percentage of positive cells for Troponin T2 (TNNT2) determined by flow cytometry in the 6 different differentiations of wild-type (WT) iPSCs, used for these experiments.

Larger scale confocal pictures of immunostained tissues at different heights basal layer(left) and cardiac ring (right) at 40X magnification: vimentin is stained in green, troponin in red, and DAPI in blue. Scale bars: 50 μm.

Evolution of contraction strain ϵA through time after seeding at days 1, 3, 7, 10 and 14. Contraction strain changes through time are significant (p<0.0001 - ANOVA for repeated measures - D1: n=57, D3: n=59 D7: n=47, D10: n=43, D14: n=36 tissues, from 3 differentiations). Contraction strain for each concentration is compared to contraction strain at day 1 (****: p<0.0001).