Generation of functional murine cardiac myocytes from induced pluripotent stem cells

Abstract

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), alpha-myosin heavy chain (alpha-MHC), and cardiac troponin T in differentiation cultures of iPS cells. Immunocytology confirmed expression of cardiomyocyte-typical proteins including sarcomeric alpha-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 beta- 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.