Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (iPSCs) as well as engineered heart muscles offer great potential for regenerative applications by CM transplantation, for the study of cardiac development and disease modeling, as well as for drug discovery and cardiotoxicity screenings in a human physiologically relevant model system. Several optimized protocols are available to efficiently differentiate iPSCs into CMs, which possess structural and functional properties of fetal CMs. However, the current methods produce a heterogeneous population of ventricular, atrial and pacemaker-like CMs strongly limiting their field of application. The generation of homogenous populations of subtype-specific iPSC-CMs and their comprehensive phenotypic comparison is crucial for a better understanding of the predominantly cardiac subtype-restricted disease mechanisms as well as for regenerative and pharmacological applications.

The goals of this study were to develop an efficient method for the directed differentiation of human iPSCs into defined functional CM subtypes in feeder-free culture conditions and to obtain a comprehensive understanding of the molecular, cell biological and functional properties of atrial and ventricular iPSC-CMs on both single cell as well as tissue level.

On the basis of temporal modulation of canonical WNT and retinoid acid signaling throughout differentiation of iPSCs via small molecules, we were able to guide the cardiac progenitor cells towards distinct cell fates resulting in homogeneous populations of either atrial or ventricular CMs. Transcriptome and proteome profiling as well as functional analysis of the CM subtypes via optical action potential screening, calcium imaging as well as engineered heart muscles demonstrate that atrial and ventricular iPSC-CMs highly correspond to CMs from the human atrium and ventricle, respectively.

In summary, this study provides a comprehensive understanding of the molecular and functional characteristics of atrial and ventricular iPSC-CMs and supports the suitability of these cells for its application in more precise disease modeling, drug screening as well as for cell-based therapeutic approaches.