Gsk3β inhibitor chir99021

Mouse embryonic stem cell line E14TG2a (mES cells) was cultured with the N2B27 base medium supplemented with 1 mM glutamine (Invitrogen), 1% nonessential amino acids (Invitrogen), 0.15 mM 1-thioglycerol (Sigma), 100 U/ml of penicillin–streptomycin (Invitrogen), 25 μg/ml of BSA (Sigma), 1 μM MEK inhibitor PD0325901 (Selleck Chemicals), 3 μM GSK3β inhibitor CHIR99021 (Selleck Chemicals), 2% KOSR (Thermo Fisher), and 1000 U/ml of ESGRO leukemia inhibitory factor LIF (Millipore) on plates coated with 0.2% gelatin.

Protocol was adapted from Lian et al. [40 (link)] with some modifications as we previously reported [32 (link)]. Briefly, hiPSCs were grown until 70–90% confluence in six-well plates. Culture media was replaced with the differentiation medium RPMI 1640 + B-27™ Supplement (without insulin) (Thermo Fisher Scientific, USA) + 0.6 mM L-ascorbic acid 2-phosphate (Sigma-Aldrich, USA), designated as RPMI/B27^-IN, and GSK3β inhibitor CHIR99021 (Selleckchem, USA) 4-12 μM was added [32 (link)]. The differentiation medium was changed after 24 h in order to remove/reduce CHIR concentration to less than 1.5 μM. Wnt inhibitor IWR-1 (Selleckchem, USA) 2.5 μM was added on day 2 [32 (link)] and was removed during the medium change on day 5. Cultures were maintained in the differentiation medium with the medium changed every 24 h. Cells were incubated at 37 °C in a humidified atmosphere with 5% CO₂. All small molecules were solubilized in DMSO (Sigma-Aldrich, USA). Cardiac markers: NK2 Homeobox 5 (NKX2–5), Troponin T and Myosin light chain 2a (MLC2a); endodermal marker hepatocyte nuclear factor 4 alpha (HNF4a; and mesodermal marker (CD44) were measured by flow cytometry on day 14 differentiation.