Human iPSC-derived cardiomyocytes and tissue engineering strategies for disease modeling and drug screening

Abstract

Improved methodologies for modeling cardiac disease phenotypes and accurately screening the efficacy and toxicity of potential therapeutic compounds are actively being sought to advance drug development and improve disease modeling capabilities. To that end, much recent effort has been devoted to the development of novel engineered biomimetic cardiac tissue platforms that accurately recapitulate the structure and function of the human myocardium. Within the field of cardiac engineering, induced pluripotent stem cells (iPSCs) are an exciting tool that offer the potential to advance the current state of the art, as they are derived from somatic cells, enabling the development of personalized medical strategies and patient specific disease models. Here we review different aspects of iPSC-based cardiac engineering technologies. We highlight methods for producing iPSC-derived cardiomyocytes (iPSC-CMs) and discuss their application to compound efficacy/toxicity screening and in vitro modeling of prevalent cardiac diseases. Special attention is paid to the application of micro- and nano-engineering techniques for the development of novel iPSC-CM based platforms and their potential to advance current preclinical screening modalities.

Keywords: Cardiac differentiation; Disease modeling; Drug screening; Induced pluripotent stem cells; Tissue engineering.

Figure 1. 2D and 3D technologies for high throughput cardiotoxicity screening

Drug dose response studies can be performed based on cardiac contractility platforms. A. Images of a muscular thin film (MTF) platform incorporating flexible cantilevers supporting cultured cardiomyocytes. Measurement of changes in cantilever position between diastole (i) and during peak systole (ii) enables quantification of force generated in cultured cardiomyocytes. Scale bar = 1 mm. Reprinted with permission (McCain, Sheehy, 2013). B. A three-dimensional filamentous model of human cardiac tissue (top left) used for analysis of contractile function through measurement of filament displacement. In this image, both cardiomyocytes and myofibroblasts were stained for expression of SM22 to show cell distribution throughout the construct. Confocal images of cardiomyocytes aligned on a single fiber show advanced sarcomere structure stained by sarcomeric α-actinin and intercellular gap junctions stained by connexin 43 (white arrows, top right). Confocal images for iPSC-CMs growing on the middle layer of a filamentous matrix and aligned along the fiber direction (bottom left). In such constructs, the formation of 3D cardiac tissue was quantified by cell number on the middle layer relative to the fiber number, and matrices with 50 μm fiber spacing were found to result in the highest value of cell per fiber (bottom right). Reprinted with permission (Ma, Koo, 2014). C. Human engineered cardiac tissues (hECTs) have been shown to mimic key aspects of the newborn human heart and thus allow functional testing for drug screening purposes. Such model systems usually incorporate a culture mold with some form of integrated endposts (top). The illustrated example shows an attached hECT on a mold from a side view. Analysis of post deflection, taking into account post length and stiffness, facilitates measurement of contractile force produced by the engineered tissue. An image is also provided showing a longitudinal section of an hECT stained with hematoxylin and eosin after 12 days in culture (bottom). Reprinted with permission (Cashman et al., 2016). D. Representative field potential waveforms collected from iPSC-CM monolayers using microelectrode arrays (MEAs) and details of the parameters extracted from the raw signal and the time averaged signal. Such analysis can be used to study drug-induced changes in cardiac electrophysiological function for prediction of compound efficacy and toxicity. Reprinted with permission (Gilchrist et al., 2015).

Figure 2. Characterization of iPSCs derived from patients with genetic cardiomyopathies for use in disease modeling applications

A. Long Q-T syndrome (LQTS) cardiomyopathy modeling. Action potentials were recorded from control and LQTS human iPSC-CMs with ventricular-like and atrial-like morphologies. The graphs indicated prolonged action potential duration (APD) – to reach 50%, 70%, and 90% of repolarization – in both ventricular-like and atrial-like LQTS iPSC-CMs when compared to control. Reprinted with permission (Itzhaki, Maizels, 2011). B. Immunofluorescence staining for desmin (DES: green) and cardiac Troponin-T (red) performed in control iPSC-CMs transduced with wild-type desmin (WT-DES: top), mutant desmin (A285V-DES: middle) and vector alone (bottom) using lentivirus. Forced expression of the mutant A285V-DES in control iPSC-CMs produced a phenotype that included diffuse isolated aggregations of desmin-positive protein (green, middle right) as observed in iPSC-CMs from dilated cardiomyopathy patients, suggesting that A285V-DES exerts a dominant-negative effect. Reprinted with permission (Tse, Ho, 2013). C. Sarcomere organization of patient-derived Barth syndrome cardiomyopathy and control iPSC-CMs. Sarcomere organization was tested in the indicated culture medium and after transfection with the indicated modified RNA. P < 0.05 vs: *, Barth syndrome cells + nGFP modified RNA, in galactose supplemented medium; #, Barth syndrome cells + nGFP modified RNA, in glucose supplemented medium. The presented data suggest treatment with TAZ modified RNA restores sarcomere regularity. Reprinted with permission (Wang, McCain, 2014). D. Cardiomyocytes derived from normal and DMD iPSCs and probed with antibodies against dystrophin (green), together with cardiac specific protein Nkx2-5 or sarcomeric α-actinin (red). Dystrophin staining was demonstrated in normal cardiomyocytes, but not in cardiomyocytes derived from DMD patients. E. Western blot for full length dystrophin detected in the lysates from normal iPSC-CMs (lane 2) and human heart tissue (lane 3) but absent in the lysate from DMD patient iPSC-CMs (lane 1). Molecular weight markers (in kDa) are shown on the left. D and E reprinted with permission (Guan, Mack, 2014).

Figure 3. Bioengineering of the myocardial niche

A. Low magnification image of a glass coverslip patterned with nanogrooves (top). Cross-sectional scanning electron microscopy (SEM) of the same surface illustrating the nanopatterned substratum (bottom). Reprinted with permission (Kim, Lipke, 2010). B. Optical mapping data show anisotropic propagation of action potentials in monolayers cultured on nanofabricated substrata, and isotropic propagation in cells cultured on flat surfaces. Point stimulation at 3 Hz was applied to the center of the cardiac monolayers as indicated by white arrows at 0 ms. The green arrow indicates the alignment of cardiomyocytes on the patterned substrates. Reprinted with permission (Kim, Lipke, 2010). C. Human stem cell-derived cardiomyocytes on flat (left), and nanopatterned (right) substrates highlighting the improvement in structural alignment in cells maintained on suitable topographies. Cells were stained for actin (green), actinin (red), and nuclei (blue). Scale bars: 50 μm. Reprinted with permission (Macadangdang et al., 2014). D. Cardiac constructs maintained without (control), and with (stimulated) electrical stimulation. (Left) Hematoxylin and Eosin (H&E) staining of unstimulated engineered tissue constructs and constructs stimulated with monophasic square wave pulses of 3 V amplitude, 3 Hz frequency and 2 ms duration. Scale bar = 1 mm. (Right) Transmission electron microscopy images of the tissues presented in the H&E images, with insets of sarcomeres. Scale bar = 2 µm in main image, 500 nm in inset. Reprinted with permission (Tandon, Marsano, 2011). E. Ultrastructural analysis of engineered cardiac biowires highlights that electrical stimulation at 6 Hz induces cardiomyocyte self-organization. Representative images of non-stimulated (control), electrically stimulated biowires illustrate sarcomere structure (Sarcomere panel: white bar; Z disks, black arrow; H zones, white arrows; m, mitochondria), and presence of desmosomes (Desmosomes panel, white arrows). Scale bar = 1 μm. Reprinted with permission. (Nunes, Miklas, 2013). F. Morphometric analysis (average ± s.d.) showing ratio of H zones to sarcomeres (CTRL vs. 6 Hz, P = 0.005), ratio of I bands to Z disks (CTRL vs. 3 Hz, P = 0.01; CTRL vs. 6 Hz, P = 0.003), and number of desmosomes per membrane length (CTRL vs. 6 Hz, P = 0.0003). In normal adult cells, the ratio of H zones to sarcomeres is 1 and the ratio of I bands to Z disks is 2. Reprinted with permission (Nunes, Miklas, 2013).