Two dimensional electrophysiological characterization of human pluripotent stem cell-derived cardiomyocyte system - PubMed (original) (raw)
Two dimensional electrophysiological characterization of human pluripotent stem cell-derived cardiomyocyte system
Huanqi Zhu et al. Sci Rep. 2017.
Abstract
Stem cell-derived cardiomyocytes provide a promising tool for human developmental biology, regenerative therapies, disease modeling, and drug discovery. As human pluripotent stem cell-derived cardiomyocytes remain functionally fetal-type, close monitoring of electrophysiological maturation is critical for their further application to biology and translation. However, to date, electrophysiological analyses of stem cell-derived cardiomyocytes has largely been limited by biologically undefined factors including 3D nature of embryoid body, sera from animals, and the feeder cells isolated from mouse. Large variability in the aforementioned systems leads to uncontrollable and irreproducible results, making conclusive studies difficult. In this report, a chemically-defined differentiation regimen and a monolayer cell culture technique was combined with multielectrode arrays for accurate, real-time, and flexible measurement of electrophysiological parameters in translation-ready human cardiomyocytes. Consistent with their natural counterpart, amplitude and dV/dtmax of field potential progressively increased during the course of maturation. Monolayer culture allowed for the identification of pacemaking cells using the multielectrode array platform and thereby the estimation of conduction velocity, which gradually increased during the differentiation of cardiomyocytes. Thus, the electrophysiological maturation of the human pluripotent stem cell-derived cardiomyocytes in our system recapitulates in vivo development. This system provides a versatile biological tool to analyze human heart development, disease mechanisms, and the efficacy/toxicity of chemicals.
Conflict of interest statement
N.N. is Chief Advisor and shareholder of a stem cell-related start-up company, Stem Cell & Device Laboratory, Inc. He is also a shareholder of ReproCELL, Inc.
Figures
Figure 1
(a) mRNA relative expression over the time course of hESC differentiation toward cardiac lineages. The value is standardized to the peak value of the time points for each gene. (b) Immunofluorescent staining for α-actinin in red showing over 90% are positive for α-actinin (scale bar = 100 um) (c) Morphological changes in hESC-CMs over the time course. Representative optical images of hESC-CMs on MEA on Day 20 and Day 35 (fluorescent staining: α-Actinin in green, nucleus in blue, scale bar = 10 um). (d) mRNA relative expression by qPCR standardized to the expression of day0 over the time course of cardiac differentiation/maturation.
Figure 2
(a) Microelectrode arrays (MEA) with 120 integrated TiN electrodes (30 μm diameter, 200 μm inter-electrode spacing) were used to culture the 2D hESC-CMs. (b) Representative optical image of hESC-CMs on top of MEA. (c) Field potential signals were detected in electrodes which are in touch with the hESC-CMs.
Figure 3
(a) Field potential features such as beat interval, field potential duration, amplitude, and local activation time (LAT) were extracted out of the signal sequences. On average, 100 out of 120 channels were selected to calculate the beat interval after the peak detection and experiments were performed in triplicate. (b) The beat interval was stable up to day 28–30 with a beat interval around 2 s. Starting from day 28, the beat interval of hESC-CMs became unstable and irregular. The standard deviation (δ) of beat interval agreed with the beat interval trend. (c) The field potential duration (FPD) ranged from 0.2 s to 0.5 s and was relatively stable throughout the differentiation process. (d) The corrected FPD ( = FPD/[beat interval/1000]1/3; Fridericia’s formula) was also relatively stable ranging from 0.2 to 0.3.
Figure 4
Peak mean amplitude (a–c) and mean beat interval (d–f) of field potential of monolayer cardiomyocytes cultured in the presence of (a,d) E4031 (K+ channel blocker), (b,e) TTX (tetrodotoxin, Na+ channel blocker) and (c,f) Nifedipine (Ca2+ channel blocker). p-values are calculated by one-way ANOVA. Data are representative of at least 2 biological replicates.
Figure 5. Field potential propagation, pacemaking and conduction velocity of the 2D hESC-CMs.
(a–h) shows a representative activation map from day 19, 20, 21, 22, 25, 26, 28, 29, respectively. The activation map was consistent over the 20-minute recording window showing a stable pace making source, but the propagation map changed over days. (i) On day 23, a contour graph of the propagation wave was generated from the activation map. (j) The center of the propagation wavelet was identified by fitting the wavelet with a circle. The origin of the circle represented the location of the pacemaking cells. The pacemaking cells were located at the edge of the MEA.
Figure 6
(a) The field potential amplitude rapidly increased until day 23 and declined at day 27. The turning point agreed with that of the beat interval. (b) The field potential amplitude upstroke speed increased from 15.3 μV/ms at day 18 to 122.9 μV/ms at day 26 and declined at day 27. (c) Conduction velocity was calculated at each day. There was an increment of conduction velocity as the development of hESC-CMs. Results from two samples were consistent.
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