Durante, M.

Frieß, J.

Helm, A.

Hessel, P.

Materna, M.

Mäntele, W.

Pignalosa, D.

Ritter, S.

Thielemann, C.

GSI Repository

GSITemplate2007 First electrophysiological studies on mouse embryonic stem cell-derived cardio-myocytes  M. Materna1,2, A. Helm1, P. Hessel1, J. Frieß3, C. Thielemann3, D. Pignalosa1, W. Mäntele2,                 M. Durante1,4 and S. Ritter1,# 1GSI, Darmstadt, Germany; 2 Goethe University Frankfurt/Main, Germany; 3University of Applied Sciences Aschaffen-burg, Germany; 4TU Darmstadt, Darmstadt, GermanyThere is emerging epidemiological evidence of an in-creased risk of adverse cardiovascular effects at low or moderate doses of ionizing radiation occurring many years after the exposure [1]. However, essentially no in-formation is available on the potential cardiovascular ef-fects associated with the exposure to heavy ions. To ad-dress this issue, we recently established in our laboratory an effective protocol for generating functional (i.e. beat-ing) cardiomyocytes from mouse embryonic stem cells (mESC). In the present study, first electrophysiological measurements were performed employing microelectrode array chips (MEA). Briefly, commercially available mESC (line D3) were differentiated by means of the hanging drop method [2]. ESC aggregate and form embryoid bodies (EBs). Seven days after initiation of the differentiation process, EBs were plated onto MEAs (1 EB/MEA). The MEAs used have a field with 60 electrodes arranged in an 8x8 grid. Each electrode has a diameter of 30 µm and the inter-electrode distance spans 200 µm. The system allows non-invasive electrophysiological measurements. At day 10, EBs start beating, pointing to the formation of cardio-myocyte-networks. In general, the number of beating EBs increased during the subsequent days and declined around day 20. Western Blot analysis and histological staining of cardio-specific markers confirmed the formation of true cardiomyocytes. Accordingly, electrophysiological meas-urements were conducted between day 10 and 20. Electrophysiological activity of untreated control samples was measured for 60 sec at a sampling rate of 10 kHz. The obtained data was then analysed with the MATLAB based program DrCell developed at the University of Ap-plied Sciences Aschaffenburg [3]. The recorded data were analysed in terms of signal amplitude, shape and beat rate. Additionally, the origin of the signal, its spreading direc-tion and velocity was registered and the number of active electrodes was determined. Representative recordings of the signal spreading, number of active electrodes and beat rate are shown in figures 1-3. Detailed analysis of the data revealed that the cell system is highly variable; large differences were observed both within a sample and between samples. This is illustrated in figure 2 and 3, where changes in the number of active electrodes and in the beat rate are depicted for 2 EBs. Based on these results we conclude that mESC-derived cardiomyocytes are an unsuitable model system for study-ing the electrophysiological responses of cardiac cells after radiation exposure which are expected to be small.    Fig. 1: Pseudocolor plot showing the delay time (ms) of action-potentials. One EB was seeded per MEA chip and 10 days after initiation of the differentiation electrical activity of cardiomyo-cytes was measured. Dark blue regions indicate the origin of the signal. Circles are electrodes.      Fig. 2: Number of active electrodes during the observation pe-riod (day 10 to day 20). The graphs display recordings from 2 EBs.    Fig.3. Beat rate of rhythmically contracting clusters in 2 EBs. References [1] Little et al., Radiat Environ Biophys, 2009, 49(2), 139-53  [2] Helm et al., GSI Scientific Report 2010, 455 [3] Nick et al., IEEE Trans. on TBME, submitted 2013 #s.ritter@gsi.de *Funded by Euratom 7th Framework Programme (no.295823),    Verein für Förderung der Tumortherapie mit schweren Ionen       e.V. and HGS-HIRe. GSI SCIENTIFIC REPORT 2012 HEALTH-17441

First electrophysiological studies on mouse embryonic stem cell-derived cardiomyocytes

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First electrophysiological studies on mouse embryonic stem cell-derived cardiomyocytes

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