Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC151
Spontaneous differentiation of embryonic stem cells leads to line-dependent functional diversity of cardiac myocytes
T. Hannes2,1, M. Khalil2, M. Wolff2, J. Hescheler3, M. Halbach4
1. DPAG, University of Oxford, Oxford, United Kingdom. 2. Paediatric Cardiology, University of Cologne, Cologne, Germany. 3. Institute for Neurophysiology, University of Cologne, Cologne, Germany. 4. Department III of Internal Medicine, University of Cologne, Cologne, Germany.
Introduction: Pluripotent stem cell-derived cardiomyocytes are thought to be an in vitro model for the early heart development and in drug testing and hold great potential for cellular cardiomyoplasty. They have been shown to resemble different subtypes of cardiomyocytes in regard of their electrophysiological properties. So far, it is not clear to which extent different embryonic stem cell lines are comparable in their quality of differentiation into cardiomyocytes. Methods: Two different transgenic stem cell lines (CGR8/AMPIGX-7, Doss 2007) and D3/aPIG44, Kolossov 2005) were grown on feeder cells in the presence of leukaemia inhibiting factor and differentiated by the hanging drop method. In order to obtain alpha-myosin heavy chain positive cardiomyocytes at a high degree of purity, puromycin was added. After 14 days of differentiation, transmembrane potentials were recorded with microelectrodes at 37°C. Results: Without purification, action potential frequency in D3/aPIG44 (n=45) was markedly higher than in CGR8/AMPIGX-7 (n=20) (7.7±0.1 Hz vs. 1.7±0.2 Hz, p<0.001). The action potential of D3/aPIG44 was markedly shorter (APD20: 7±0.1 Hz vs. 36±5 Hz, APD50: 14.6±0.2 Hz vs. 80.6±10.2 Hz, APD90: 29±0.4 Hz vs 188.9±13.2 Hz, all p<0.001). Comparison of purified (n=28) and non-purified (n=45) D3/aPIG44 showed a slightly smaller frequency of purified ESC-CM clusters (7.2±0.1 Hz vs. 7.7±0.1 Hz, p<0.01) and an increase in APD20 (7±0.2 Hz vs. 8.4±0.4 Hz, p<0.05. and in APD90 (20±0.4 Hz vs. 29.6±0.4 Hz, p<0.05). In contrast, purified CGR8/AMPGIX-7 (n=20) had a strong decrease of APDs with purification compared to unpurified CGR8/AMPIGX-7 (APD20: 26.3±3.1 Hz vs. 36±5 Hz, APD50: 50.1±6.1 Hz vs. 80.6±10.2 Hz, APD90: 79.8±7.3 Hz vs. 188.98±13.2 Hz, all p<0.05). The frequency did not significantly change. Conclusions: The spontaneous differentiation of D3/aPIG44 and CGR8/AMPIGX-7 leads to electrophysiologically distinct cardiomyocyte phaenotypes in regard of beating frequency, APD and effect of purification under control of the alpha-myosin heavy chain-promoter. CGR8/AMPIGX-7 strongly resemble the ventricular phaenotype, while D3/aPIG44 share many properties with sino-atrial cells. This questions the comparability and validity of the spontaneous differentiation of embryonic stem cells as a model for cardiac development and drug testing and underlines the necessity of unraveling factors to control cardiac subtype differentiation.
Where applicable, experiments conform with Society ethical requirements