Rpmi1640 3 knockout serum replacement

Before the initiation of cardiomyocytes differentiation, hiPSCs were dissociated into clumps using Dispase (Sigma-Aldrich) and were plated onto Matrigel Basement Membrane Matrix Growth Factor Reduced (BD Biosciences). When the cells attained a confluence of 90–100% (depending on the cell line), mTeSR was replaced with CDM medium containing 10 μM CHIR99021 (Sigma-Aldrich) for 24 h. The medium was then replaced with CDM medium containing 5 ng/mL bFGF (Peprotech) for 1 day, followed by CDM medium with or without the saponin⁺ compound, then cultured for another day. Next, the cultures were changed to half of the CDM medium containing 5 μM IWP2 (Sigma-Aldrich) with or without the saponin⁺ compound for 2 days. Finally, the differentiated cells were cultured in RPMI1640/3% KnockOut™ Serum Replacement (Gibco) medium with or without the saponin⁺ compound for further growth. The medium was changed every 48 h. On Days 10, 11, 13, and 15, the medium was replaced with glucose-free DMEM (Invitrogen) containing 4 mmol/L L-lactic acid (Sigma) to metabolically select and purify cardiomyocytes (hiPS-CMs). 15 days after starting the differentiation, the optimally induced hiPS-CMs exhibited spontaneous contraction were used for transplantation into mice.

Human iPSCs were grown until 100% confluent on Matrigel (BD Biosciences). For the next 24 h, mTeSR was substituted with CDM medium containing 10 μM CHIR99021 (Sigma‐Aldrich). The medium was then changed to a CDM medium containing 5 ng/ml bFGF (Peprotech) for 24 h, followed by another 24 h of culture in a CDM medium containing the saponin⁺ compound. For the next two days, half of the culture medium was changed with CDM medium containing 5 μM IWP2 (Sigma‐Aldrich) with the saponin⁺ compound. Finally, the differentiated cells were cultured in RPMI 1640/3% KnockOut Serum Replacement (Gibco) medium with the saponin⁺ compound for 48 h before being transferred to a fresh medium. The protocol is shown in Figure S1B. On Days 8, 9, 11, 13, and 15, the medium was replaced with glucose‐free DMEM (Invitrogen) containing 4 mmol/L L‐lactic acids (Sigma) to metabolically select and purify cardiomyocytes. The saponin⁺ compound‐induced hiPSC‐CMs expressed cardiac‐specific markers (Figure S1C), exhibited spontaneous contraction (supporting movie), and were used for transplantation.