Berend van Meer

Three-dimensional cardiac microtissues composed of cardiomyocytes and endothelial cells co-differentiated from human pluripotent stem cells

ABSTRACT Cardiomyocytes and endothelial cells in the heart are in close proximity and in constant dialogue. Endothelium regulates the size of the heart, supplies oxygen to the myocardium and secretes factors that support cardiomyocyte function. Robust and predictive cardiac disease models that faithfully recapitulate native human physiology in vitro would therefore ideally incorporate this cardiomyocyte-endothelium crosstalk. Here, we have generated and characterized human cardiac microtissues in vitro that integrate both cell types in complex 3D structures. We established conditions for simultaneous differentiation of cardiomyocytes and endothelial cells from human pluripotent stem cells following initial cardiac mesoderm induction. The endothelial cells expressed cardiac markers that were also present in primary cardiac microvasculature, suggesting cardiac endothelium identity. These cell populations were further enriched based on surface markers expression, then recombined allowing development of beating 3D structures termed cardiac microtissues. This in vitro model was robustly reproducible in both embryonic and induced pluripotent stem cells. It thus represents an advanced human stem cell-based platform for cardiovascular disease modelling and testing of relevant drugs. Summary: Co-differentiation of endothelial cells and cardiomyocytes from human pluripotent stem cells provides a cardiac microtissue model with potential applications for disease modelling and drug discovery.

Concise Review: Measuring Physiological Responses of Human Pluripotent Stem Cell Derived Cardiomyocytes to Drugs and Disease

Cardiomyocytes from human pluripotent stem cells (hPSC) are of growing interest as models to understand mechanisms underlying genetic disease, identify potential drug targets and for safety pharmacology as they may predict human relevant effects more accurately and inexpensively than animals or other cell models. Crucial to their optimal use are accurate methods to quantify cardiomyocyte phenotypes accurately and reproducibly. Here, we review current methods for determining biophysical parameters of hPSC‐derived cardiomyocytes (hPSC‐CMs) that recapitulate disease and drug responses. Even though hPSC‐CMs as currently available are immature, various biophysical methods are nevertheless already providing useful insights into the biology of the human heart and its maladies. Advantages and limitations of assays currently available looking toward applications of hPSC‐CMs are described with examples of how they have been used to date. This will help guide the choice of biophysical method to characterize healthy cardiomyocytes and their pathologies in vitro. Stem Cells 2016;34:2008–2015

Measuring physiological responses of human pluripotent stem cell derived cardiomyocytes to drugs and disease

Cardiomyocytes from human pluripotent stem cells (hPSC) are of growing interest as models to understand mechanisms underlying genetic disease, identify potential drug targets and for safety pharmacology as they may predict human relevant effects more accurately and inexpensively than animals or other cell models. Crucial to their optimal use are accurate methods to quantify cardiomyocyte phenotypes accurately and reproducibly. Here, we review current methods for determining biophysical parameters of hPSC‐derived cardiomyocytes (hPSC‐CMs) that recapitulate disease and drug responses. Even though hPSC‐CMs as currently available are immature, various biophysical methods are nevertheless already providing useful insights into the biology of the human heart and its maladies. Advantages and limitations of assays currently available looking toward applications of hPSC‐CMs are described with examples of how they have been used to date. This will help guide the choice of biophysical method to characterize healthy cardiomyocytes and their pathologies in vitro. Stem Cells 2016;34:2008–2015

Cytostretch, an Organ-on-Chip Platform

Organ-on-Chips (OOCs) are micro-fabricated devices which are used to culture cells in order to mimic functional units of human organs. The devices are designed to simulate the physiological environment of tissues in vivo. Cells in some types of OOCs can be stimulated in situ by electrical and/or mechanical actuators. These actuations can mimic physiological conditions in real tissue and may include fluid or air flow, or cyclic stretch and strain as they occur in the lung and heart. These conditions similarly affect cultured cells and may influence their ability to respond appropriately to physiological or pathological stimuli. To date, most focus has been on devices specifically designed to culture just one functional unit of a specific organ: lung alveoli, kidney nephrons or blood vessels, for example. In contrast, the modular Cytostretch membrane platform described here allows OOCs to be customized to different OOC applications. The platform utilizes silicon-based micro-fabrication techniques that allow low-cost, high-volume manufacturing. We describe the platform concept and its modules developed to date. Membrane variants include membranes with (i) through-membrane pores that allow biological signaling molecules to pass between two different tissue compartments; (ii) a stretchable micro-electrode array for electrical monitoring and stimulation; (iii) micro-patterning to promote cell alignment; and (iv) strain gauges to measure changes in substrate stress. This paper presents the fabrication and the proof of functionality for each module of the Cytostretch membrane. The assessment of each additional module demonstrate that a wide range of OOCs can be achieved.

Altered calcium handling and increased contraction force in human embryonic stem cell derived cardiomyocytes following short term dexamethasone exposure

One limitation in using human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) for disease modeling and cardiac safety pharmacology is their immature functional phenotype compared with adult cardiomyocytes. Here, we report that treatment of human embryonic stem cell derived cardiomyocytes (hESC-CMs) with dexamethasone, a synthetic glucocorticoid, activated glucocorticoid signaling which in turn improved their calcium handling properties and contractility. L-type calcium current and action potential properties were not affected by dexamethasone but significantly faster calcium decay, increased forces of contraction and sarcomeric lengths, were observed in hESC-CMs after dexamethasone exposure. Activating the glucocorticoid pathway can thus contribute to mediating hPSC-CMs maturation.

MUSCLEMOTION: a versatile open software tool to quantify cardiomyocyte and cardiac muscle contraction in vitro and in vivo

Rationale: There are several methods to measure cardiomyocyte and muscle contraction, but these require customized hardware, expensive apparatus, and advanced informatics or can only be used in single experimental models. Consequently, data and techniques have been difficult to reproduce across models and laboratories, analysis is time consuming, and only specialist researchers can quantify data. Objective: Here, we describe and validate an automated, open-source software tool (MUSCLEMOTION) adaptable for use with standard laboratory and clinical imaging equipment that enables quantitative analysis of normal cardiac contraction, disease phenotypes, and pharmacological responses. Methods and Results: MUSCLEMOTION allowed rapid and easy measurement of movement from high-speed movies in (1) 1-dimensional in vitro models, such as isolated adult and human pluripotent stem cell-derived cardiomyocytes; (2) 2-dimensional in vitro models, such as beating cardiomyocyte monolayers or small clusters of human pluripotent stem cell-derived cardiomyocytes; (3) 3-dimensional multicellular in vitro or in vivo contractile tissues, such as cardiac “organoids,” engineered heart tissues, and zebrafish and human hearts. MUSCLEMOTION was effective under different recording conditions (bright-field microscopy with simultaneous patch-clamp recording, phase contrast microscopy, and traction force microscopy). Outcomes were virtually identical to the current gold standards for contraction measurement, such as optical flow, post deflection, edge-detection systems, or manual analyses. Finally, we used the algorithm to quantify contraction in in vitro and in vivo arrhythmia models and to measure pharmacological responses. Conclusions: Using a single open-source method for processing video recordings, we obtained reliable pharmacological data and measures of cardiac disease phenotype in experimental cell, animal, and human models.

Fabrication and Characterization of an Upside-Down Carbon Nanotube Microelectrode Array

Microelectrode arrays (MEAs) are widely used in biological application to locally stimulate and record the electrical activity of living cells. Here, a novel fabrication process for a carbon nanotube (CNT)-based MEA integrated on the backside of a free standing stretchable membrane is reported. The new process flow overcomes the manually intensive procedures used in the previous works. The microfabricated upside-down CNT MEA consists of microelectrodes with an area of 110

Quantification of Muscle Contraction In Vitro and In Vivo Using MUSCLEMOTION Software: From Stem Cell-Derived Cardiomyocytes to Zebrafish and Human Hearts

\mu \text{m}^{2}

Versatile open software to quantify cardiomyocyte and cardiac muscle contraction in vitro and in vivo

covered with cobalt-grown CNTs. The surface area enhancement and the foam-like morphology of the CNTs allow an increase of the charge injection per unit area at the electrode-electrolyte interface, resulting in a significantly lower electrochemical impedance of the electrodes. In particular, at 1 kHz, the fabricated CNT-MEA electrodes show a reduction of the overall impedance up to 96% in comparison with benchmark TiN electrodes. The obtained results confirm the effectiveness of the proposed surface texturing through CNT integration. Moreover, the quality and the morphology as well as the biocompatibility of the fabricated CNT-based electrodes were assessed. The obtained results demonstrate that significant improvement can be achieved by integrating structured nanoporous material on MEAs.

InPulse CrackIT Challenge: Development of An In Vitro Platform Using Physiologically Mature Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes to Screen for Drug-Induced Effects on Cardiac Contractility

Quantification of contraction is essential to the study of cardiac diseases, injury, and responses to drugs. While there are many techniques to assess contractility, most rely on costly, dedicated hardware and advanced informatics, and can only be used in specific experimental models. We have developed an automated open‐source software tool (MUSCLEMOTION) for use with standard imaging equipment, to assess contractility in vitro and in vivo and quantify responses to drugs and diseases. We describe high‐speed and disturbance‐free acquisition of images from either electrically paced or non‐paced human pluripotent stem cell‐derived cardiomyocytes, isolated adult cardiomyocytes, zebrafish hearts, and human echocardiograms. Recordings are then used as input for automated batch analysis by the MUSCLEMOTION software tool configured with specific settings and parameters tailored to the recording technique. Details on accuracy, interpretation, and troubleshooting are discussed. Acquisition duration depends on the experimental setup and aim, but quantification of drug or disease responses in an in vitro muscle model can typically be completed within a few hours.