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(Circulation. 2008;118:507-517.)
© 2008 American Heart Association, Inc.

Molecular Cardiology

Generation of Functional Murine Cardiac Myocytes From Induced Pluripotent Stem Cells

Christina Mauritz, DVM; Kristin Schwanke, PhD; Michael Reppel, MD; Stefan Neef, MD; Katherina Katsirntaki; Lars S. Maier, MD; Filomain Nguemo, PhD; Sandra Menke; Moritz Haustein; Juergen Hescheler, MD; Gerd Hasenfuss, MD; Ulrich Martin, PhD

From the Leibniz Research Laboratories for Biotechnology and Artificial Organs, Department of Cardiac, Thoracic, Transplantation, and Vascular Surgery, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany (C.M., K.S., K.K., S.M., U.M.); Institute of Neurophysiology, University of Cologne, Cologne, Germany (M.R., F.N., M.H., J.H.); Department of Cardiology, University of Luebeck, Luebeck, Germany (M.R.); and Department of Cardiology and Pneumology, University Medical Center Goettingen, Goettingen, Germany (S.N., L.S.M., G.H.).

Correspondence to Ulrich Martin, PhD, Leibniz Research Laboratories for Biotechnology and Artificial Organs, Carl-Neuberg Strasse 1, 30625 Hannover, Germany. E-mail martin.ulrich{at}mh-hannover.de

Received March 10, 2008; accepted May 12, 2008.

Background— The recent breakthrough in the generation of induced pluripotent stem (iPS) cells, which are almost indistinguishable from embryonic stem (ES) cells, facilitates the generation of murine disease– and human patient–specific stem cell lines. The aim of this study was to characterize the cardiac differentiation potential of a murine iPS cell clone in comparison to a well-established murine ES cell line.

Methods and Results— With the use of a standard embryoid body–based differentiation protocol for ES cells, iPS cells as well as ES cells were differentiated for 24 days. Although the analyzed iPS cell clone showed a delayed and less efficient formation of beating embryoid bodies compared with the ES cell line, the differentiation resulted in an average of 55% of spontaneously contracting iPS cell embryoid bodies. Analyses on molecular, structural, and functional levels demonstrated that iPS cell–derived cardiomyocytes show typical features of ES cell–derived cardiomyocytes. Reverse transcription polymerase chain reaction analyses demonstrated expression of marker genes typical for mesoderm, cardiac mesoderm, and cardiomyocytes including Brachyury, mesoderm posterior factor 1 (Mesp1), friend of GATA2 (FOG-2), GATA-binding protein 4 (GATA4), NK2 transcription factor related, locus 5 (Nkx2.5), T-box 5 (Tbx5), T-box 20 (Tbx20), atrial natriuretic factor (ANF), myosin light chain 2 atrial transcripts (MLC2a), myosin light chain 2 ventricular transcripts (MLC2v), -myosin heavy chain (-MHC), and cardiac troponin T in differentiation cultures of iPS cells. Immunocytology confirmed expression of cardiomyocyte-typical proteins including sarcomeric -actinin, titin, cardiac troponin T, MLC2v, and connexin 43. iPS cell cardiomyocytes displayed spontaneous rhythmic intracellular Ca²⁺ fluctuations with amplitudes of Ca²⁺ transients comparable to ES cell cardiomyocytes. Simultaneous Ca²⁺ release within clusters of iPS cell–derived cardiomyocytes indicated functional coupling of the cells. Electrophysiological studies with multielectrode arrays demonstrated functionality and presence of the β-adrenergic and muscarinic signaling cascade in these cells.

Conclusions— iPS cells differentiate into functional cardiomyocytes. In contrast to ES cells, iPS cells allow derivation of autologous functional cardiomyocytes for cellular cardiomyoplasty and myocardial tissue engineering.

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CLINICAL PERSPECTIVE

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