In: Stem Cell Research & Therapy, 8 (2017), Nr. 229. S. 1-15. ISSN 1757-6512
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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.
Dokumententyp: | Artikel |
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Titel der Zeitschrift: | Stem Cell Research & Therapy |
Band: | 8 |
Nummer: | 229 |
Verlag: | BioMed Central |
Ort der Veröffentlichung: | London |
Erstellungsdatum: | 24 Okt. 2017 11:40 |
Erscheinungsjahr: | 2017 |
ISSN: | 1757-6512 |
Seitenbereich: | S. 1-15 |
Institute/Einrichtungen: | Zentrale und Sonstige Einrichtungen > Deutsches Krebsforschungszentrum
Zentrale und Sonstige Einrichtungen > Max-Planck-Institute allgemein > MPI fuer Med. Forschung Medizinische Fakultät Heidelberg und Uniklinikum > Medizinische Universitäts-Klinik und Poliklinik Medizinische Fakultät Heidelberg und Uniklinikum > Institut fuer Physiologie und Pathophysiologie |
DDC-Sachgruppe: | 610 Medizin |