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Stem Cell Res Ther. 2017 Oct 16;8(1):229. doi: 10.1186/s13287-017-0681-4.

Subtype-specific differentiation of cardiac pacemaker cell clusters from human induced pluripotent stem cells.

Author information

1
Department of Cardiology, Medical University Hospital Heidelberg, INF 410, D-69120, Heidelberg, Germany. patrick.schweizer@med.uni-heidelberg.de.
2
DZHK (German Centre for Cardiovascular Research), partner site Heidelberg/Mannheim, University of Heidelberg, INF 410, D-69120, Heidelberg, Germany. patrick.schweizer@med.uni-heidelberg.de.
3
Department of Cardiology, Medical University Hospital Heidelberg, INF 410, D-69120, Heidelberg, Germany.
4
DZHK (German Centre for Cardiovascular Research), partner site Heidelberg/Mannheim, University of Heidelberg, INF 410, D-69120, Heidelberg, Germany.
5
Institute of Physiology and Pathophysiology, Division of Cardiovascular Physiology, Heidelberg University, INF 326, D-69120, Heidelberg, Germany.
6
Institute of Physiology and Pathophysiology, Division of Neuro- and Sensory Physiology, Heidelberg University, INF 326, D-69120, Heidelberg, Germany.
7
Department of Cell and Developmental Biology, Max-Planck-Institute for Molecular Biomedicine, Röntgenstrasse, 20, D-48149, Münster, Germany.
8
Unit Tumor Models, German Cancer Research Center (DKFZ), Heidelberg, INF 280, D-69120, Heidelberg, Germany.
9
Dermato-Oncology (G300), German Cancer Research Center (DKFZ), Heidelberg, INF 280, D-69120, Heidelberg, Germany.
10
Department of Dermatology, Venereology and Allergology, University Medical Center Mannheim, Ruprecht-Karl University of Heidelberg, Theodor-Kutzer-Ufer 1-3, D-68167, Mannheim, Germany.
11
Department of Molecular Neurobiology, Max-Planck-Institute for Medical Research, Jahnstrasse 29, D-69120, Heidelberg, Germany.

Abstract

BACKGROUND:

Human induced pluripotent stem cells (hiPSC) harbor the potential to differentiate into diverse cardiac cell types. Previous experimental efforts were primarily directed at the generation of hiPSC-derived cells with ventricular cardiomyocyte characteristics. Aiming at a straightforward approach for pacemaker cell modeling and replacement, we sought to selectively differentiate cells with nodal-type properties.

METHODS:

hiPSC were differentiated into spontaneously beating clusters by co-culturing with visceral endoderm-like cells in a serum-free medium. Subsequent culturing in a specified fetal bovine serum (FBS)-enriched cell medium produced a pacemaker-type phenotype that was studied in detail using quantitative real-time polymerase chain reaction (qRT-PCR), immunocytochemistry, and patch-clamp electrophysiology. Further investigations comprised pharmacological stimulations and co-culturing with neonatal cardiomyocytes.

RESULTS:

hiPSC co-cultured in a serum-free medium with the visceral endoderm-like cell line END-2 produced spontaneously beating clusters after 10-12 days of culture. The pacemaker-specific genes HCN4, TBX3, and TBX18 were abundantly expressed at this early developmental stage, while levels of sarcomeric gene products remained low. We observed that working-type cardiomyogenic differentiation can be suppressed by transfer of early clusters into a FBS-enriched cell medium immediately after beating onset. After 6 weeks under these conditions, sinoatrial node (SAN) hallmark genes remained at high levels, while working-type myocardial transcripts (NKX2.5, TBX5) were low. Clusters were characterized by regular activity and robust beating rates (70-90 beats/min) and were triggered by spontaneous Ca2+ transients recapitulating calcium clock properties of genuine pacemaker cells. They were responsive to adrenergic/cholinergic stimulation and able to pace neonatal rat ventricular myocytes in co-culture experiments. Action potential (AP) measurements of cells individualized from clusters exhibited nodal-type (63.4%) and atrial-type (36.6%) AP morphologies, while ventricular AP configurations were not observed.

CONCLUSION:

We provide a novel culture media-based, transgene-free approach for targeted generation of hiPSC-derived pacemaker-type cells that grow in clusters and offer the potential for disease modeling, drug testing, and individualized cell-based replacement therapy of the SAN.

KEYWORDS:

Automaticity; Differentiation; Pacemaker; Pluripotent stem cells

PMID:
29037217
PMCID:
PMC5644063
DOI:
10.1186/s13287-017-0681-4
[Indexed for MEDLINE]
Free PMC Article

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