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1.
J Tissue Eng ; 13: 20417314221127908, 2022.
Article in English | MEDLINE | ID: mdl-36277058

ABSTRACT

Paucity of physiologically relevant cardiac models has limited the widespread application of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes in drug development. Here, we performed comprehensive characterization of hiPSC-derived cardiomyocyte subtypes from 2D and 3D cultures and established a novel 3D model to study impulse initiation and propagation. Directed differentiation approaches were used to generate sinoatrial nodal (SANCM), atrial (ACM) and ventricular cardiomyocytes (VCM). Single cell RNA sequencing established that the protocols yield distinct cell populations in line with expected identities, which was also confirmed by electrophysiological characterization. In 3D EHT cultures of all subtypes, we observed prominent expression of stretch-responsive genes such as NPPA. Response to rate modulating drugs noradrenaline, carbachol and ivabradine were comparable in single cells and EHTs. Differences in the speed of impulse propagation between the subtypes were more pronounced in EHTs compared with 2D monolayers owing to a progressive increase in conduction velocities in atrial and ventricular cardiomyocytes, in line with a more mature phenotype. In a novel binary EHT model of pacemaker-atrial interface, the SANCM end of the tissue consistently paced the EHTs under baseline conditions, which was inhibited by ivabradine. Taken together, our data provide comprehensive insights into molecular and electrophysiological properties of hiPSC-derived cardiomyocyte subtypes, facilitating the creation of next generation composite cardiac models for drug discovery, disease modeling and cell-based regenerative therapies.

2.
Elife ; 112022 10 11.
Article in English | MEDLINE | ID: mdl-36217819

ABSTRACT

Each heartbeat is triggered by the sinoatrial node (SAN), the primary pacemaker of the heart. Studies in animal models have revealed that pacemaker cells share a common progenitor with the (pro)epicardium, and that the pacemaker cardiomyocytes further diversify into 'transitional', 'tail', and 'head' subtypes. However, the underlying molecular mechanisms, especially of human pacemaker cell development, are poorly understood. Here, we performed single cell RNA sequencing (scRNA-seq) and trajectory inference on human induced pluripotent stem cells (hiPSCs) differentiating to SAN-like cardiomyocytes (SANCMs) to construct a roadmap of transcriptional changes and lineage decisions. In differentiated SANCM, we identified distinct clusters that closely resemble different subpopulations of the in vivo SAN. Moreover, the presence of a side population of proepicardial cells suggested their shared ontogeny with SANCM, as also reported in vivo. Our results demonstrate that the divergence of SANCM and proepicardial lineages is determined by WNT signaling. Furthermore, we uncovered roles for TGFß and WNT signaling in the branching of transitional and head SANCM subtypes, respectively. These findings provide new insights into the molecular processes involved in human pacemaker cell differentiation, opening new avenues for complex disease modeling in vitro and inform approaches for cell therapy-based regeneration of the SAN.


Subject(s)
Induced Pluripotent Stem Cells , Animals , Cell Differentiation , Humans , Myocytes, Cardiac , Sinoatrial Node , Transforming Growth Factor beta
3.
Stem Cell Reports ; 16(11): 2589-2606, 2021 11 09.
Article in English | MEDLINE | ID: mdl-34653403

ABSTRACT

Retinoic acid (RA) signaling plays an important role during heart development in establishing anteroposterior polarity, formation of inflow and outflow tract progenitors, and growth of the ventricular compact wall. RA is also utilized as a key ingredient in protocols designed for generating cardiac cell types from pluripotent stem cells (PSCs). This review discusses the role of RA in cardiogenesis, currently available protocols that employ RA for differentiation of various cardiovascular lineages, and plausible transcriptional mechanisms underlying this fate specification. These insights will inform further development of desired cardiac cell types from human PSCs and their application in preclinical and clinical research.


Subject(s)
Cell Differentiation/physiology , Cell Lineage/physiology , Heart/physiology , Myocardium/metabolism , Pluripotent Stem Cells/metabolism , Signal Transduction/physiology , Tretinoin/metabolism , Animals , Cell Differentiation/genetics , Cell Lineage/genetics , Gene Expression Regulation, Developmental , Heart/embryology , Humans , Models, Cardiovascular , Myocardium/cytology , Pluripotent Stem Cells/cytology , Receptors, Retinoic Acid/genetics , Receptors, Retinoic Acid/metabolism , Signal Transduction/genetics , Sinoatrial Node/cytology , Sinoatrial Node/embryology , Sinoatrial Node/metabolism , T-Box Domain Proteins/genetics , T-Box Domain Proteins/metabolism
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