*2.1. Generation of hiPSC and Engineered Heart Tissue (EHT)*

The hiPSC line C25 (kind gift from Alessandra Moretti, Munich, Germany) was reprogrammed by lentivirus [1] and was expanded in FTDA medium and differentiated in a three step protocol based on growth factors and a small molecule Wnt inhibitor DS07 (kind gift from Dennis Schade, Dortmund, Germany) as previously published [14–16]. The hiPSC line ERC018 were generated in-house from skin fibroblasts of a healthy subject using the CytoTuneTM-iPS Sendai Reprogramming Kit (Thermo Fisher Scientific, Waltham, MA, USA) and differentiated to cardiomyocytes as described for C25. In brief, confluent undifferentiated cells were dissociated (0.5 mM EDTA; 10 min) and cultivated in spinner flasks (30 <sup>×</sup> 106 cells/100 mL; 40 rpm) for embryoid body formation overnight. Mesodermal differentiation was initiated in embryoid bodies over three days in suspension culture with growth factors (BMP-4 (R&D Systems, 314-BP), activin-A (R&D Systems, 338-AC) and FGF2 (PeproTech, 100-18B)). Cardiac differentiation was performed either in adhesion or in suspension culture with Wnt-inhibitor DS07. Cells were cultured in a humidified temperature and gas-controlled incubator (37 ◦C, 5% CO2, 5% O2 and 21% O2 for final cardiac differentiation). At day 14 the spontaneously beating hiPSC-CMs were dissociated with collagenase II (Worthington, LS004176; 200 U/mL, 3.5 h). For quality control, dissociated cells were analyzed by flow cytometry as described before [14,17] with anti-cardiac troponin T-FITC (Miltenyi, clone REA400, 130-112-756). All differentiation runs utilized for

this study had 64–96% cardiac Troponin T positive cells (Supplementary Materials Figure S2). Further characterization of the non-CMs within the EHT was evaluated previously by our group [6], showing low expression of vimentin-positive fibroblast-like markers and the virtual absence of endothelial, neuronal, and endodermal markers. ICell and iCell<sup>2</sup> cardiomyocytes are commercially available hiPSC-CM lines purchased from Fujifilm Cellular Dynamics (Madison, Wisconsin, USA) and were included in expression analysis after culture in EHT. For three-dimensional culture EHTs were generated with 1 <sup>×</sup> 106 hiPSC-CM/<sup>100</sup> <sup>μ</sup>L EHT mastermix as previously described [18]. EHTs were cultured in a 37 ◦C, 7% CO2 and 40% O2 humidified cell culture incubator with a medium consisting of DMEM (F0415, Biochrom; Berlin, Germany), 10% heat-inactivated horse serum (Gibco 26050, Thermo Fisher Scientific, Waltham, MA, USA), 1% penicillin/streptomycin (Gibco 15140), insulin (10 μg/mL; Sigma-Aldrich I9278, St. Louis, MO, USA) and aprotinin (33 μg/mL; Sigma-Aldrich A1153). EHTs were cultured for at least 3 weeks to allow maturation. The work with hiPSC was approved by the Ethical Committee of the University Medical Center Hamburg-Eppendorf (Az. PV4798, 28.10.2014). All donors gave written informed consent.

#### *2.2. Human Adult Heart Tissue*

This investigation conforms to all principles outlined by the Declaration of Helsinki and the Medical Association of Hamburg. All materials from patients were taken with informed consent of the donors. Left ventricular free wall and left ventricular septum samples were obtained from patients undergoing heart transplantation or from aortic valve surgery.
