I0161

Human embryonic stem cells (hESCs), line H7, were cultured in serum-free mTeSR1 medium (Stemcell Technologies, #85852) on Matrigel-coated culture plates and passaged when cells reached suitable confluence.
Cardiomyocyte differentiation was carried out when cells reached 85–95% confluence. For differentiation, cells were cultured with RPMI/B-27 medium. Gsk3 inhibitor CHIR-99021(12 mM, Selleck, S2924) or a WNT inhibitor IWR-1 (5 mM, Sigma, I0161) was added to the cells on day 0 and 2–3, respectively. On day 4–6, cells were treated with fresh RPMI/B-27 medium. Contracting cells appeared between 7 and 9 days post differentiation and the hESC-derived cardiomyocytes after ≥ 25 days differentiation were used in the experiments. The successful differentiation of myocardial cells can be determined by the appearance of beating cells, as the beating cells and three-dimensional cell bulge can be observed in the plates when the induced differentiation goes well.

For feeder-free cjiPSC culture, the cjiPSCs (C6 and C10) were cultured on xeno-free recombinant Laminin-511 E8 fragment-coated dishes (TAKARA, iMatrix-511silk) with PluriSTEM Human ES/iPS cell media (Sigma-Aldrich, SCM130). The cells were passaged approximately every 6–7 days as clumps after treatment with 0.5 mM EDTA in PBS for 10 min. For OF culture, the cjiPSCs were cultured with DK20F20 (DMEM/F12 [Thermo Fisher, 11320-033] supplemented with 20% (vol/vol) KSR, 1 mM sodium pyruvate [Thermo Fisher, 11360-070], 2 mM GlutaMax [Thermo Fisher, 35050061], 0.1 mM NEAA, 0.1 mM 2-mercaptoethanol [Thermo Fisher, 21985-023], penicillin-streptomycin at 25 U/ml [Thermo Fisher, 15070063], and recombinant human bFGF) at 20 ng/ml on Mitomycin C-treated MEFs (2.5×10⁵ cells/well of a six-well plate). For OF/IWR1 culture, IWR1 was added at 2.5 µM (Sigma, I0161). For single-cell passage, cells were dissociated into single-cell suspension every 6–7 days with Accutase (Sigma-Aldrich, A6964) and seeded at a density of 1×10⁵ cells/9 cm². Culture medium was supplemented with 10 µM ROCK inhibitor (Tocris, 1254) until 24 hr after passage.

All the chemical inhibitors were diluted in distilled water except for AG1478 [T4182, SIGMA-ALDRICH, in dimethyl sulfoxide (DMSO)] and LMT-28 (SML1628, SIGMA-ALDRICH, in methanol). Therefore, we used either 0.1% DMSO or 0.1% methanol as negative controls of AG1478 or LMT-28 treatments. AG1478 was added at 3 μM to the medium right after tail amputation. To disrupt different signaling pathways during Mtz ablation, we treated 3-dpf larvae with designated drugs and Mtz. After neuromast ablation, the chemical inhibitors were either washed out with Mtz or added back to be kept in the medium. The COX inhibitor diclofenac sodium salt (D6899, SIGMA-ALDRICH), a specific blocker of neutrophil recruitment, was used at 3 μM. The TNFα inhibitor pentoxifylline (PTX, P1784, SIGMA-ALDRICH) was used at 35 μM. The IL-6 inhibitors LMT-28 and the Wnt/β-catenin inhibitor IWR-1 (I0161, SIGMA-ALDRICH) were used at 10 μM.