eprintid: 23646
rev_number: 16
eprint_status: archive
userid: 1589
dir: disk0/00/02/36/46
datestamp: 2017-10-24 11:40:02
lastmod: 2024-03-09 08:36:25
status_changed: 2017-10-24 11:40:02
type: article
metadata_visibility: show
creators_name: Schweizer, Patrick A.
creators_name: Darche, Fabrice F.
creators_name: Ullrich, Nina D.
creators_name: Geschwill, Pascal
creators_name: Greber, Boris
creators_name: Rivinius, Rasmus
creators_name: Seyler, Claudia
creators_name: Müller-Decker, Karin
creators_name: Draguhn, Andreas
creators_name: Utikal, Jochen
creators_name: Koenen, Michael
creators_name: Katus, Hugo A.
creators_name: Thomas, Dierk
title: Subtype-specific differentiation of cardiac pacemaker cell clusters from human induced pluripotent stem cells
subjects: 610
divisions: 850300
divisions: 851330
divisions: 910100
divisions: 53200
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.
date: 2017
publisher: BioMed Central
id_scheme: DOI
ppn_swb: 1656729113
own_urn: urn:nbn:de:bsz:16-heidok-236463
language: eng
bibsort: SCHWEIZERPSUBTYPESPE2017
full_text_status: public
publication: Stem Cell Research & Therapy
volume: 8
number: 229
place_of_pub: London
pagerange: 1-15
issn: 1757-6512
citation:   Schweizer, Patrick A. ; Darche, Fabrice F. ; Ullrich, Nina D. ; Geschwill, Pascal ; Greber, Boris ; Rivinius, Rasmus ; Seyler, Claudia ; Müller-Decker, Karin ; Draguhn, Andreas ; Utikal, Jochen ; Koenen, Michael ; Katus, Hugo A. ; Thomas, Dierk  (2017) Subtype-specific differentiation of cardiac pacemaker cell clusters from human induced pluripotent stem cells.  Stem Cell Research & Therapy, 8 (229).  pp. 1-15.  ISSN 1757-6512     
document_url: https://archiv.ub.uni-heidelberg.de/volltextserver/23646/1/13287_2017_Article_681.pdf