Directed Cardiomyocyte Differentiation of hiPS Cells

Human iPS cells were differentiated into CMs using the established matrix sandwich method^{35 (link)} with modifications. Briefly, 6 days before initiating differentiation, hiPS cell colonies were passaged on human embryonic stem cell–qualified Matrigel-coated plates (0.05 mg/mL; BD Biosciences) using Gentle Cell Dissociation Buffer (Stemcell Technologies) and cultured as a monolayer in mTeSR1 with 1× Y-27632 ROCK inhibitor (Stemcell Technologies) in a normal oxygen atmosphere. When cells reached 80% confluence, cold mTeSR1 with Growth Factor Reduced Matrigel (0.033 mg/mL) was added to create an overlay of Matrigel. Differentiation was initiated 24 hours later (day 0) by culturing the cells in RPMI-1640 medium (Life Technologies) supplemented with B27 (without insulin; Life Technologies), 100 ng/mL activin A (Miltenyi Biotec), and 10 ng/mL FGF2 (Miltenyi Biotec) for 24 hours. On the next day, the medium was replaced by RPMI-1640 medium supplemented with B27 without insulin, 10 ng/mL BMP4 (Miltenyi Biotec), and 5 ng/mL FGF2 for 4 days. By day 5, cells were cultured in RPMI-1640 medium supplemented with B27 complete (Life Technologies) and 1% NEAA and changed every 2 to 3 days.