Monolayer-based cardiac differentiation and cardiac enrichment by sphere formation

Human iPSK3 cells were seeded on 24-well plates coated with Geltrex (Life Technologies) at a cell density of 4 × 10⁵ cells/well. Cells were maintained in mTeSR serum-free medium with 10 µM ROCK inhibitor (ROCKi) Y27632 (Sigma) for the first day and in mTeSR medium only for another four days. The cardiomyocytes differentiation was induced by modulating Wnt pathways with small molecules CHIR99021 (a Wnt activator) and IWP4 (a Wnt inhibitor) (Giwi protocol)¹¹. To optimize the protocol, the treatment conditions of CHIR99021 (StemCell Technologies, Inc.) were investigated. Briefly, cells were cultivated in RPMI plus 2% B27 serum-free supplement minus insulin medium (Life Technologies) with 5 µM or 10 µM CHIR99021 (day 0) for 24 hours or 48 hours and then CHIR99021 was withdrawn from the medium. After another two days, the cells were then cultivated in the medium with 5 µM IWP4 (Stemgent) for two days. At day 6 or 7, the medium was changed to RRMI plus 2% B27 and beating cells were observed at day 12–14. Based on the analysis of expression of cardiac markers, the optimized condition for CHIR99021 induction was 10 µM and 48 hours treatment.

Cardiac differentiation was also induced with growth factors. Undifferentiated iPSK3 cells were treated with 100 ng/mL recombinant human activin A (R&D Systems) in RPMI/B27-insulin medium (day 0). On day 1, the cells were maintained in RPMI/B27-insulin medium supplemented with 10 ng/mL recombinant human bone morphogenetic protein 4 (BMP-4; R&D Systems). Four days later (i.e., on day 5), the medium was switched to RPMI/B27 and subsequently the cells were fed every alternative day with this medium until harvest^7,14. To enhance cardiomyocyte differentiation, the day 15 cells in monolayer (2D) were harvested and cultured in Ultra-Low Attachment (ULA) 24-well plate (Corning) for cardiac sphere formation.