Deep phenotyping of human induced pluripotent stem cell–derived atrial and ventricular cardiomyocytes

Abstract

Generation of homogeneous populations of subtype-specific cardiomyocytes (CMs) derived from human induced pluripotent stem cells (iPSCs) and their comprehensive phenotyping is crucial for a better understanding of the subtype-related disease mechanisms and as tools for the development of chamber-specific drugs. The goals of this study were to apply a simple and efficient method for differentiation of iPSCs into defined functional CM subtypes in feeder-free conditions and to obtain a comprehensive understanding of the molecular, cell biological, and functional properties of atrial and ventricular iPSC-CMs on both the single-cell and engineered heart muscle (EHM) level. By a stage-specific activation of retinoic acid signaling in monolayer-based and well-defined culture, we showed that cardiac progenitors can be directed towards a highly homogeneous population of atrial CMs. By combining the transcriptome and proteome profiling of the iPSC-CM subtypes with functional characterizations via optical action potential and calcium imaging, and with contractile analyses in EHM, we demonstrated that atrial and ventricular iPSC-CMs and -EHM highly correspond to the atrial and ventricular heart muscle, respectively. This study provides a comprehensive understanding of the molecular and functional identities characteristic of atrial and ventricular iPSC-CMs and -EHM and supports their suitability in disease modeling and chamber-specific drug screening.

The Emergence of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs) as a Platform to Model Arrhythmogenic Diseases

“…The resulting iPSCs are then differentiated into cardiomyocytes through exposure to a variety of stimuli [20][21][22] and culture media [22,23]. In most cases, around 90% of cells are cardiac troponin 2 (TNNT2)-positive [24,25]. Although hiPSC differentiation protocols usually generate a large proportion of ventricular-like hiPSC-CMs [23], a mixed population of ventricular-like, atrial-like, and sinoatrial node-like cells are typically produced [26][27][28][29].…”

“…In the context of disease modeling, it is essential to develop differentiation protocols that generate homogeneous populations of subtype specific cardiomyocytes to determine cell specific disease phenotypes. Current protocols effectively differentiate iPSCs toward the ventricular cell subtype [24,25,30]. However, more efficient and reproducible differentiation protocols to generate the atrial cell subtype have been described [25].…”

“…Current protocols effectively differentiate iPSCs toward the ventricular cell subtype [24,25,30]. However, more efficient and reproducible differentiation protocols to generate the atrial cell subtype have been described [25]. A protocol based on the activation of the retinoic acid signaling cascade to initiate atrial cardiomyocyte differentiation generates over 85% of the atrial subtype [25].…”

Stem Cell Therapy of Myocardial Infarction: A Promising Opportunity in Bioengineering

“…Although many protocols have been developed to efficiently generate hPSC‐CMs with high yield and purity, most of them result in heterogeneous CM subtypes consisting predominantly of those with ventricular characteristics with a small amount of atrial‐like and nodal‐like cells . Substantial evidence indicates that RA plays a role in regulating fate specification of atrial versus ventricular CMs, and it can be used to generate 85–95% hESC‐atrial CMs . Studies on early cardiac developmental stages reveal that the specification of ventricular and atrial CMs is dependent on induction of the appropriate mesoderm .…”

“…Substantial evidence indicates that RA plays a role in regulating fate specification of atrial versus ventricular CMs, and it can be used to generate 85–95% hESC‐atrial CMs . Studies on early cardiac developmental stages reveal that the specification of ventricular and atrial CMs is dependent on induction of the appropriate mesoderm . RA specifies that the RAHDL2+ mesoderm produces atrial CMs and the CD235a+ mesoderm gives rise to ventricular CMs .…”