Functional video-based analysis of 3D cardiac structures generated from human embryonic stem cells

Abstract

Human embryonic stem cells (hESCs) differentiated into cardiomyocytes (CM) often develop into complex 3D structures that are composed of various cardiac cell types. Conventional methods to study the electrophysiology of cardiac cells are patch clamp and microelectrode array (MEAs) analyses. However, these methods are not suitable to investigate the contractile features of 3D cardiac clusters that detach from the surface of the culture dishes during differentiation. To overcome this problem, we developed a video-based motion detection software relying on the optical flow by Farnebäck that we call cBRA (cardiac beat rate analyzer). The beating characteristics of the differentiated cardiac clusters were calculated based on the local displacement between two subsequent images. Two differentiation protocols, which profoundly differ in the morphology of cardiac clusters generated and in the expression of cardiac markers, were used and the resulting CM were characterized. Despite these differences, beat rates and beating variabilities could be reliably determined using cBRA. Likewise, stimulation of β-adrenoreceptors by isoproterenol could easily be identified in the hESC-derived CM. Since even subtle changes in the beating features are detectable, this method is suitable for high throughput cardiotoxicity screenings.

User-Friendly and Parallelized Generation of Human Induced Pluripotent Stem Cell-Derived Microtissues in a Centrifugal Heart-on-a-Chip

“…This specifically tailored software is based on optical flow motion tracking algorithms similar to previous approaches. 13,30,31 The analysis yields a time-dependent displacement vector field, which serves to characterize and compare beating patterns of individual tissues. The obtained time-dependent vector field may be represented as a quiver plot that can be used as an overlay to the raw video recording to visualize the beating behavior more clearly (Fig.…”

A Human 3D Cardiomyocyte Risk Model to Study the Cardiotoxic Influence of X-rays and Other Noxae in Adults

“…To measure the clinical relevant functionality of the contraction, optical techniques often rely on fluorescent protein dyes for calcium or voltage sensing, but loading target cells with dye might alter the electrophysiological properties and potentially bias results [37]. We made use of dye-free video analyses, presented in [22]. By doing so, we could demonstrate that radiation induces subtle changes in CM function by increasing the beat rate in a mostly dose-dependent manner and decreasing rhythmicity.…”

“…At the time of transfer, hPSC-CMC were at least 100 days old and maintained in insulin-free medium. To determine the beat rate and rhythmicity of individual hPSC-CMC, the software cBRA (cardiac Beat Rate Analyzer) and the setup presented by Nitsch and colleagues [22] were used. For measurements of external stimuli, the temperature of the microscope-mounted heated plate was adjusted in the range of 32-38 °C (recordings of n = 18 individual hPSC-CMC), or medium spiked with 1 µM each of isoprenaline (Merck, #I6504, n = 15 individual hPSC-CMC), propranolol (Merck, #P0884, n = 15) or verapamil (Merck, #V4629, n = 7) was consecutively added to controls (n = 15) in the order mentioned.…”

Human-induced pluripotent stem cell-derived cardiomyocytes, 3D cardiac structures, and heart-on-a-chip as tools for drug research

“…Daily et al [ 41 ] established an optimized protocol of 3D CMs cultures preparation, cell loading protocol and measurement registration by a plate reader, facilitating precise action potential detection. Nitsch et al, in turn, proposed an optical video-based measurements of contraction properties of 3D structures [ 132 ].…”