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Cardiac Myocytes Derived from Murine Reprogrammed Fibroblasts: Intact Hormonal Regulation, Cardiac Ion Channel Expression and Development of ContractilityPfannkuche K.1,* · Liang H.1,* · Hannes T.2,* · Xi J.1,* · Fatima A.1 · Nguemo F.1 · Matzkies M.1 · Wernig M.4 · Jaenisch R.4,5 · Pillekamp F.2 · Halbach M.1 · Schunkert H.3 · Šaric T.1,# · Hescheler J.1,# · Reppel M.1,3,#
1Institute for Neurophysiology, University of Cologne,2Pediatric Cardiology, University of Cologne,3Department of Cardiology, Medical University of Lübeck,4Whitehead Institute for Biomedical Research and5Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts,*Authors contributed equally to the manuscript,#corresponding authors Corresponding Author
Jürgen Hescheler, Tomo Saric, Michael Reppel
Institut für Neurophysiologie, Universität zu Köln
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Aims: Induced pluripotent stem (iPS) cells have a developmental potential similar to that of blastocyst-derived embryonic stem (ES) cells and may serve as an autologous source of cells for tissue repair, in vitro disease modelling and toxicity assays. Here we aimed at generating iPS cell-derived cardiomyocytes (CMs) and comparing their molecular and functional characteristics with CMs derived from native murine ES cells. Methods and Results: Beating cardiomyocytes were generated using a mass culture system from murine N10 and O9 iPS cells as well as R1 and D3 ES cells. Transcripts of the mesoderm specification factor T-brachyury and non-atrial cardiac specific genes were expressed in differentiating iPS EBs. Using immunocytochemistry to determine the expression and intracellular organisation of cardiac specific structural proteins we demonstrate strong similarity between iPS-CMs and ES-CMs. In line with a previous study electrophysiological analyses showed that hormonal response to β-adrenergic and muscarinic receptor stimulation was intact. Action potential (AP) recordings suggested that most iPS-CMs measured up to day 23 of differentiation are of ventricular-like type. Application of lidocaine, Cs+, SEA0400 and verapamil+ nifedipine to plated iPS-EBs during multi-electrode array (MEA) measurements of extracellular field potentials and intracellular sharp electrode recordings of APs revealed the presence of INa, If, INCX, and ICaL, respectively, and suggested their involvement in cardiac pacemaking, with ICaL being of major importance. Furthermore, iPS-CMs developed and conferred force to avitalized ventricular tissue that was responsive to β-adrenergic stimulation. Conclusions: Our data demonstrate that the cardiogenic potential of iPS cells is comparable to that of ES cells and that iPS-CMs possess all fundamental functional elements of a typical cardiac cell, including spontaneous beating, hormonal regulation, cardiac ion channel expression and contractility. Therefore, iPS-CMs can be regarded as a potentially valuable source of cells for in vitro studies and cellular cardiomyoplasty.
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