Induced Pluripotent Stem Cell Protocol

Control and TS iPSC lines were generated using retroviral infection with pMXs-SOX2, pMXs-OCT3/4, pMXs-MYC and pMXs-KLF4 expression plasmids (Add-gene) generated by Dr. Shinya Yamanaka’s group20 (link). The iPSCs were cultured on irradiated DR4 mouse embryonic fibroblast feeders using standard ES media with 10–15 ng/ml bFGF (R & D Systems), and cells were passaged with dispase (3 unit/ml, Invitrogen). The G1216A in exon 8a was detected by sequencing of PCR products from DNA harvested from fibroblasts and iPSC lines using primers for human Cav1.2 exon 8a. Immunocytochemistry, RT-PCR, microarray, karyotyping and teratoma formation assay were performed using standard protocols. For in vitro generation of cardiomyocytes, embryoid bodies were cultured with Wnt3a (100 ng/ml, R&D Systems) 27 (link). Whole-cell patch clamp recordings in single cardiomyocytes were conducted using standard methods. Live cell Ca²⁺ imaging was performed in single cardiomyocytes loaded with 5 μM Fluo-4 AM and 0.02% Pluronic F-127 (Molecular Probes) using fast line scanning (1.92 ms/line) on a confocal microscope (LSM 510 Meta, Carl Zeiss) with a 63× lens (NA=1.4). R-roscovitine was obtained from Sigma-Aldrich.