To differentiate human hiPSC into cardiac lineages, we used a well-defined protocol. (Lian et al., 2012 (link); Lian et al., 2013 (link)). Briefly, hiPSC were plated at day -4 of differentiation on Matrigel GFR diluted at 1:100 at 125,000 cells/cm2 and were cultured in mTESR medium (STEMCELL Technologies) supplemented with Pen-Strep, which was changed daily until day 0. From day 0 to day 7 cells were cultured in RPMI medium (Lonza) supplemented with B27 minus insulin and Pen-Strep (both from Gibco). At day 0 of differentiation, cells were treated with 12 µM CHIR99021 (Axon Medchem) for 24 h. On day 3, cells were treated with 5 µM IWP4 (STEMCELL technologies) for 48 h. From day 7 of differentiation onwards, cells were maintained in RPMI medium supplemented with complete B27 (Gibco) and Pen-Strep, and the medium was changed every 3 days.
CM were enriched by two rounds of metabolic selection where indicated. Briefly, on day 11 and 16, the medium was transiently changed to RPMI medium without Glucose (Gibco) supplemented with complete B27, Pen-Strep and 5 mMl -lactic acid (Sigma-Aldrich) for 72 h. From day 14–16, the resting period, cells were cultured with regular differentiation medium.
CM were enriched by two rounds of metabolic selection where indicated. Briefly, on day 11 and 16, the medium was transiently changed to RPMI medium without Glucose (Gibco) supplemented with complete B27, Pen-Strep and 5 mM