Final optimized media formulations for each of the three steps of cardiac differentiation.

The basal medium RPMI 1640 was found to be most successful for
cardiac induction during phase 2 and phase 3 and therefore was
maintained for phase 4. The addition of the thiol 1-thioglycerol was
found to be essential for hEB formation and survival. High
concentrations of PVA enhanced hiPSC hEB formation. Insulin was
essential for hEB formation. The combination of BMP4 and FGF2 was
optimal for mesoderm specification whilst FBS or human serum was
essential in phase 3 for <90% contracting hEB
differentiation efficiency. The addition of lipids in phase 2 and 4
enhanced hEB survival. A low concentration of Y27632 (ROCK
inhibitor) enhanced the reproducibility of hEB formation. The
xeno-/serum-free variant of the phase 3 medium required the addition
of HSA and L-ascorbic acid to maintain cardiac differentiation and
produces ∼60% of hEB contracting by d15 of
differentiation.