Cryopreservation of Human Embryonic Stem Cells Derived-Cardiomyocytes Induced by BMP2 in Serum-Free Condition

Although previous studies showed that cardiomyocytes (CMs) can be generated from human embryonic stem cells (hESCs), the protocols for cryopreservation of hESC-derived CMs is not available to date. Here, we report on the efficient generation of hESC-derived CMs by direct differentiation using BMP2 in a serum-free condition, along with successful cryopreservation of derived CMs using Rho-associated kinase (ROCK) inhibitor. To induce differentiation, hESCs were treated with activin A and BMP2 for 5 days. A mesodermal gene, Brachyury, was expressed from day 3, and cardiac-specific markers such as Nkx2.5 and cTnI were detected at day 14. Furthermore, these cardiac progenitors expressed ion channel-related transcripts such as HCN1 and HCN2 from day 10. Beating clusters were observed from 14 days of differentiation for up to 35 days. Using mass cryopreservation, we froze hESC-derived CMs at 2 stages, at day 12 and 16 (prebeating and postbeating), after treating with ROCK inhibitor, Y-27632. Postthaw survival of CMs was higher in day 12 group compared to day 16, and some cell clusters from day 12 group recovered their contraction. From transmission electron microscope (TEM) analysis, less ultrastructural alterations were observed in day 12 group. Our results provide an insight into the use of BMP2 for cardiac lineage differentiation in a serum-free condition and a possibility of long-term storage of hESC-derived CMs.