3D Cardiac Differentiation

In order to generate cardiac embryoid bodies (cEBs), hPSCs were incubated with 1 mg/mL of collagenase I (Sigma-Aldrich) in PBS with Ca++ and Mg++ supplemented with 20% FBS for 20 minutes at 37 °C. Afterwards, cells were detached to form small aggregates of 10 to 20 cells by 0.05% Trypsin-EDTA (Thermo Fisher Scientific) digestion and mechanical scrapping. Aggregates were plated in ultra-low attachment plates (Corning) at 37 °C and 5% CO2²with the following basal culture medium: StemPro®-34 SFM (Life Technologies), 1% glutamine, 1% penicillin–streptomycin, 150 μg/mL transferrin (Roche Life Sciences), 0.039 μL/mL monothioglycerol (Sigma-Aldrich), 50 μg/mL ascorbic acid (Sigma-Aldrich). To induce differentiation, the basal culture medium was supplemented with BMP4 (Tocris), bFGF (Thermo Fisher Scientific), Activin A (Tocris), and the Wnt/B-catenin inhibitor XAV939 (Tocris). At day 15 the percentage of NKX2-5/GFP(+) cells were analyzed by flow cytometry. Again, these cardiomyocytes shows spontaneous beating activity²⁸.