Cardiomyocyte development from human induced pluripotent stem cells (iPSC)

Induced-pluripotent stem cell-derived cardiomyocyte (IPSC-CM) models can improve understanding of pathophysiology through disease modelling and can be used for cardiotoxicity screening of drugs, ultimately reducing reliance on animal models; however these models have not been extensively functionally characterised, which was the aim of this study. A contracting IPSC-CM model was generated and characterised for atrial and ventricle-specific cardiac gene expression with RNAsequencing. These data are to be correlated with functional assays. Cryopreserved IPSCs were recovered from liquid nitrogen and thawed into a 6 ml volume of StemFlexTM supplemented with 10 µM rho-associated protein kinase (ROCK) inhibitor (Y-27632 dihydrochloride; Sigma). IPSCs were collected by centrifugation at 100 x g for 90 seconds and the cell pellet gently resuspended in StemFlexTM, before being plated onto vitronectin-coated tissue culture plates. Spontaneous differentiation of IPSCs was initiated by replacement of StemFlexTM medium with DMEM/F-12 supplemented with 10% (vol/vol) FBS. Medium changes were performed every 3-4 days for up to 15 days. RNA was purified from monolayer cell culture using RNeasy Mini Kit (Qiagen, Manchester, UK) according to the manufacturer’s instructions. Data was the sequenced on a NovaSeq with paired-end protocol.