Cardiac Organoid Generation from hiPSCs

hiPSC-based cardiac organoids were generated by modifying the protocol previously published (Lewis-Israeli et al., 2021 (link)). Briefly, hiPSC aggregates were formed by seeding hiPSCs at 10,000 cells/well on ultra-low attachment V-shaped 96-well plate (Sbio, MS-9096-VZ), previously rinsed with anti-adherence rinse solution (Stem Cell Technology, 07010). The plate was then centrifuged at 100 g for 3 min and placed in the CO2 incubator (day −2). After 24 h, 80 µL of media was changed to fresh Essential 8 medium. On day 0, media was changed to RPMI 1640 (Welgene, LM 011-01) based differentiation media supplemented with B27 minus insulin (Gibco, A1517001) containing 5 µM CHIR99021 (TOCRIS, 4,423) and incubated for 48 h. After 48 h (day 2), media was changed to RPMI-1640 + B27 minus insulin differentiation media containing 2 µM Wnt-C59 (Selleckchem, S7037) and incubated for 48 h. On day 4, the media was changed to fresh RPMI-1640 + B27 minus insulin differentiation media. From day 6 to day 30, the media was replenished with fresh RPMI-1640 + B27 with insulin (Gibco, 17504-44) differentiation media every other day. Spontaneously beating organoids start arising from day 9. Contractility measurement was performed at day 30. The organoids were generated from the C2N3 iPSC line (Figures 2, 3) and the SCVI-111 (Figure 5).