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Functional characterization of cardiomyocytes derived from murine induced pluripotent stem cells in vitro

Alexey Kuzmenkin^*,1, Huamin Liang^*,,,1, Guoxing Xu^*,,, Kurt Pfannkuche^*, Hardy Eichhorn^*, Azra Fatima^*, Hongyan Luo^*,,, Tomo ari^*,, Marius Wernig^¶,2, Rudolf Jaenisch^¶,|| and Juergen Hescheler^*,,3

^* Institute for Neurophysiology, Medical Center, and

Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany;

Department of Physiology and

Chinese-German Stem Cell Center, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; and

^¶ Whitehead Institute for Biomedical Research and

^|| Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

³ Correspondence: Institute for Neurophysiology, Medical Center, University of Cologne, Robert-Koch-Str. 39, 50931 Cologne, Germany. E-mail: j.hescheler{at}uni-koeln.de

Several types of terminally differentiated somatic cells can be reprogrammed into a pluripotent state by ectopic expression of Klf4, Oct3/4, Sox2, and c-Myc. Such induced pluripotent stem (iPS) cells have great potential to serve as an autologous source of cells for tissue repair. In the process of developing iPS-cell-based therapies, the major goal is to determine whether differentiated cells derived from iPS cells, such as cardiomyocytes (CMs), have the same functional properties as their physiological in vivo counterparts. Therefore, we differentiated murine iPS cells to CMs in vitro and characterized them by RT-PCR, immunocytochemistry, and electrophysiology. As key markers of cardiac lineages, transcripts for Nkx2.5, MHC, Mlc2v, and cTnT could be identified. Immunocytochemical stainings revealed the presence of organized sarcomeric actinin but the absence of mature atrial natriuretic factor. We examined characteristics and developmental changes of action potentials, as well as functional hormonal regulation and sensitivity to channel blockers. In addition, we determined expression patterns and functionality of cardiac-specific voltage-gated Na⁺, Ca²⁺, and K⁺ channels at early and late differentiation stages and compared them with CMs derived from murine embryonic stem cells (ESCs) as well as with fetal CMs. We conclude that iPS cells give rise to functional CMs in vitro, with established hormonal regulation pathways and functionally expressed cardiac ion channels; CMs generated from iPS cells have a ventricular phenotype; and cardiac development of iPS cells is delayed compared with maturation of native fetal CMs and of ESC-derived CMs. This difference may reflect the incomplete reprogramming of iPS cells and should be critically considered in further studies to clarify the suitability of the iPS model for regenerative medicine of heart disorders.—Kuzmenkin, A., Liang, H., Xu, G., Pfannkuche, K., Eichhorn, H., Fatima, A., Luo, H., ari, T., Wernig, M., Jaenisch, R., Hescheler, J. Functional characterization of cardiomyocytes derived from murine induced pluripotent stem cells in vitro.

Key Words: iPS cells • cardiac differentiation • electrophysiology • patch-clamp • reprogramming