TY  - JOUR
N2  - 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.
ID  - heidok23646
A1  - Schweizer, Patrick A.
A1  - Darche, Fabrice F.
A1  - Ullrich, Nina D.
A1  - Geschwill, Pascal
A1  - Greber, Boris
A1  - Rivinius, Rasmus
A1  - Seyler, Claudia
A1  - Müller-Decker, Karin
A1  - Draguhn, Andreas
A1  - Utikal, Jochen
A1  - Koenen, Michael
A1  - Katus, Hugo A.
A1  - Thomas, Dierk
SN  - 1757-6512
CY  - London
VL  - 8
PB  - BioMed Central
IS  - 229
AV  - public
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/23646/
TI  - Subtype-specific differentiation of cardiac pacemaker cell clusters from human induced pluripotent stem cells
SP  - 1
Y1  - 2017///
EP  - 15
JF  - Stem Cell Research & Therapy
ER  -