*2.3. hESC Culture and Di*ff*erentiation to Cardiomyocytes*

The hESCs were cultured on tissue culture dishes coated with Geltrex (Life Technologies, Thermo Fisher Scientific) in mTeSR1 culture medium (STEMCELL Technologies, Vancouver, BC, Canada) with daily media changes. The cells were passaged every 3–4 days using Accutase (STEMCELL Technologies). The undifferentiated phenotype of the hESCs was checked daily using a light microscope. In order to differentiate hESCs to cardiomyocytes, they were dissociated by Versene (Life Technologies, Thermo Fisher Scientific), resuspended in mTeSR1 + 5 μM Y27632 and seeded onto Geltrex-coated plates at a density of 3 <sup>×</sup> 105 cells/cm<sup>2</sup> and grown for the next four days with daily medium change. Following that, the cells were treated with 6 μM CHIR99021 (Selleckchem, Houston, TX, USA) in insulin-free RPMI/B27 medium (Life Technologies) for 24 h. The medium was replaced with basal medium for another 2 days. At day 3, the culture medium was subsequently replaced with 5 μM IWP-2 (Tocris Bioscience, Minneapolis, MN, USA) in insulin-free RPMI/B27 for 48 h. On day 7, the culture medium was changed to RPMI/B27 containing insulin (Life Technologies, Thermo Fisher Scientific), and the culture medium was refreshed every 3 days thereafter.

#### *2.4. Alkaline Phosphatase Staining*

Cells were washed in PBS twice, fixed with 80% alcohol for at least 2 h at 4 ◦C, followed by soaking in ddH2O for 2–3 min, and 100 mM Tris-HCl (pH 8.2–8.5) for 5 min. Alkaline phosphatase substrate working solution (Vector Laboratories, Burlingame, CA, USA) was added for 1 h and stained colonies were visualized under light microscope.

#### *2.5. Reverse Transcription-Polymerase Chain Reaction (RT-PCR)*

Total RNA was isolated with TRIzol reagent (Invitrogen, Thermo Fisher Scientific) and quantified by spectrophotometry at 260 nm. 3 μg of total RNA was reverse transcribed using SuperScript III Reverse Transcriptase (Invitrogen, Thermo Fisher Scientific) at 55 ◦C for 1 h into complementary DNA, which was then used as the template for subsequent PCR reactions. The PCR reactions were run with the following cycling conditions: 94 ◦C for 5 min, followed by 25 or 30 cycles at 94 ◦C (denaturation) for 30 s, 58–62 ◦C for 30 s (annealing), 72 ◦C for 45 s (synthesis). The primer sequences are shown in Supplementary Tables S1 and S2. Amplified RT-PCR products were analyzed on 2% agarose gels and visualized using ethidium bromide staining and SPOT camera system (Diagnostic Instruments, Sterling Height, MI, USA).
