Iwr 1

IWR-1 was obtained from ENZO (Farmingdale, NY, USA), PI3K inhibitor LY294002 was purchased from Sigma-Aldrich (St Louis, MO, USA), and recombinant human tumor necrosis factor (TNF)-α was from R&D Systems (Minneapolis, MN, USA). Primary antibodies against E-cadherin, N-cadherin, Snail, Survivin, Vimentin, p-Akt (Ser473), t-Akt, β-catenin, and β-Actin as well as secondary antibodies conjugated to horseradish peroxidase (HRP), Alexa-594, and FITC were obtained from Cell Signaling Technology (Danvers, MA, USA). Lipofectamine 2000, SYBR Green, and the total RNA purification kit were purchased from Invitrogen (Carlsbad, CA, USA), and the RT-Premix Kit was obtained from ELPIS Biotech (Daejeon, South Korea).

Human cardiac organoids were purchased from Novoheart (Hong Kong) and cultured with StemPro34 SFM (Invitrogen) medium supplemented with ascorbic acid (AA, 50 mg/mL; Sigma), 2 mM GlutaMAX-1 (Invitrogen), BMP4 (10 ng/mL), IWR-1 (5 mM; Enzo Life Sciences) and human recombinant activin-A (10 ng/mL; Invitrogen). The culture medium was replaced with the StemPro34 SFM medium with AA every two days^{70 (link)}. Cardiac organoids were disassembled using TrypLE express upon reaching confluence, and infected with shMMP14 lentivirus for 24 h. Cardiac organoids were subsequently selected with Puromycin (0.5 ug/ml) for one week.

Undifferentiated hUiPSCs were digested into smaller clusters using ACCUTASE and seeded onto Matrigel-coated plates at 3 · 10⁵ cells/10 cm² in mTeSR1 medium until they reached 80%–90% confluence. Thereafter, the cells were digested into single-cell suspensions and seeded into ultralow-attachment six-well plates (Corning) and cardiac differentiation was induced as described elsewhere[29 (link)]. Briefly, cells were kept for 24 hours in mTeSR1 medium with Matrigel (40 mg/mL), BMP4 (1 ng/mL; Invitrogen) and Rho kinase inhibitor (ROCK) (10 μM; R&D) under a hypoxic condition with 3% O₂. Then, the first group of cells was washed and replaced in cardiogenic medium (Medium C -REAC), containing: StemPro34 SFM (Invitrogen) with ascorbic acid (AA, 50 mg/mL; Sigma), 2mM Gluta-MAX-1 (Invitrogen), BMP4 (10 ng/mL), and human recombinant activin-A (10 ng/mL; Invitrogen). The second group of cells (Medium C+REAC) was replaced in the presence of the cardiogenic medium and additionally exposed to REAC for 72 hours. On day 4 both groups of cells were incubated with IWR-1, a Wnt inhibitor (5 mM; Enzo Life Sciences). On day 8, all cells were transferred to a normoxic environment and maintained in cardiogenic medium until the end of experiment (day 14). Cardiac mesodermal cells organized in functional contracting clusters were detected as early as day 8.