Deep phenotyping of human induced pluripotent stem cell–derived atrial and ventricular cardiomyocytes
Lukas Cyganek, … , Gerd Hasenfuss, Kaomei Guan
Lukas Cyganek, … , Gerd Hasenfuss, Kaomei Guan
Published June 21, 2018
Citation Information: JCI Insight. 2018;3(12):e99941. https://doi.org/10.1172/jci.insight.99941.
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Research Article Muscle biology Stem cells

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.

Authors

Lukas Cyganek, Malte Tiburcy, Karolina Sekeres, Kathleen Gerstenberg, Hanibal Bohnenberger, Christof Lenz, Sarah Henze, Michael Stauske, Gabriela Salinas, Wolfram-Hubertus Zimmermann, Gerd Hasenfuss, Kaomei Guan

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Figure 1

Directed differentiation of iPSCs into atrial or ventricular cardiomyocytes (CMs).

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Directed differentiation of iPSCs into atrial or ventricular cardiomyocy...
(A) Schematic of the defined differentiation protocols. Small molecules CHIR and IWP2, which modulate canonical WNT signaling, were applied for the induction of iPSCs into CMs. Retinoic acid (RA) was used to induce the atrial subtype specification. Metabolic selection with lactate was applied to achieve a higher purity of iPSC-CMs. (B and C) Expression of genes involved in WNT and RA signaling was assessed by reverse transcriptase PCR analysis in control and RA-treated cells. Shown are representative images (B, iPSC line iBM76.3) and semiquantitative analysis of gene expression (C, n = 2–3 independent differentiation experiments performed for each iPSC line; 3 iPSC lines were used). Results were quantified according to intensity and normalized to GAPDH expression. (D) Flow cytometry analysis of iPSC-CMs with or without lactate selection for cardiac troponin T (cTNT). Top: Representative measurements (iPSC line iBM76.3); gray peaks represent the isotype control. Bottom: Quantitative analysis (n = 11–14 control and n = 9–15 RA-treated independent differentiation experiments from 3 iPSC lines). Data are presented as mean ± SEM. **P < 0.01, ***P < 0.001 by nonparametric Mann-Whitney U test.