Jesse Macadangdang

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

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.

Isolation and Mechanical Measurements of Myofibrils from Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Summary Tension production and contractile properties are poorly characterized aspects of excitation-contraction coupling of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Previous approaches have been limited due to the small size and structural immaturity of early-stage hiPSC-CMs. We developed a substrate nanopatterning approach to produce hiPSC-CMs in culture with adult-like dimensions, T-tubule-like structures, and aligned myofibrils. We then isolated myofibrils from hiPSC-CMs and measured the tension and kinetics of activation and relaxation using a custom-built apparatus with fast solution switching. The contractile properties and ultrastructure of myofibrils more closely resembled human fetal myofibrils of similar gestational age than adult preparations. We also demonstrated the ability to study the development of contractile dysfunction of myofibrils from a patient-derived hiPSC-CM cell line carrying the familial cardiomyopathy MYH7 mutation (E848G). These methods can bring new insights to understanding cardiomyocyte maturation and developmental mechanical dysfunction of hiPSC-CMs with cardiomyopathic mutations.

Electroconductive Nanopatterned Substrates for Enhanced Myogenic Differentiation and Maturation

Electrically conductive materials provide a suitable platform for the in vitro study of excitable cells, such as skeletal muscle cells, due to their inherent conductivity and electroactivity. Here it is demonstrated that bioinspired electroconductive nanopatterned substrates enhance myogenic differentiation and maturation. The topographical cues from the highly aligned collagen bundles that form the extracellular matrix of skeletal muscle tissue are mimicked using nanopatterns created with capillary force lithography. Electron beam deposition is then utilized to conformally coat nanopatterned substrates with a thin layer of either gold or titanium to create electroconductive substrates with well-defined, large-area nanotopographical features. C2C12 cells, a myoblast cell line, are cultured for 7 d on substrates and the effects of topography and electrical conductivity on cellular morphology and myogenic differentiation are assessed. It is found that biomimetic nanotopography enhances the formation of aligned myotubes and the addition of an electroconductive coating promotes myogenic differentiation and maturation, as indicated by the upregulation of myogenic regulatory factors Myf5, MyoD, and myogenin (MyoG). These results suggest the suitability of electroconductive nanopatterned substrates as a biomimetic platform for the in vitro engineering of skeletal muscle tissue.

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide(1). Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The hearts extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale(2). Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering(3-5). A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling(2). Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart(6-9). Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)(8) and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function(10-14). Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively(15,16). Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (λ = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 °C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS(2).

NanoMEA: a versatile platform for high-throughput analysis of structure-function relationships in human stem cell-derived excitable cells and tissues

Somatic cells derived from human pluripotent stem cell (hPSC) sources hold significant potential as a means to improve current in vitro screening assays. However, their inconsistent ability to recapitulate the structural and functional characteristics of native cells has raised questions regarding their ability to accurately predict the functional behavior of human tissues when exposed to chemical or pathological insults. In addition, the lack of cytoskeletal organization within conventional culture platforms prevents analysis of how structural changes in human tissues affect functional performance. Using cation-permeable hydrogels, we describe the production of multiwell nanotopographically-patterned microelectrode arrays (nanoMEAs) for studying the effect of structural organization on hPSC-derived cardiomyocyte and neuronal function in vitro. We demonstrate that nanoscale topographic substrate cues promote the development of more ordered cardiac and neuronal monolayers while simultaneously enhancing cytoskeletal organization, protein expression patterns, and electrophysiological function in these cells. We then show that these phenotypic improvements act to alter the sensitivity of hPSC-derived cardiomyocytes to treatment with arrhythmogenic and conduction-blocking compounds that target structural features of the cardiomyocyte. Similarly, we demonstrate that neuron sensitivity to synaptic blockers is increased when cells are maintained on nanotopographically-patterned Nafion surfaces. The improved structural and functional capacity of hPSC-derived cardiomyocyte and neuronal populations maintained on nanoMEAs may have important implications for improving the predictive capabilities of cell-based electrophysiological assays used in preclinical screening applications.

Engineered developmental niche enables predictive phenotypic screening in human dystrophic cardiomyopathy

Directed differentiation of human pluripotent stem cells (hPSCs) into cardiomyocytes typically produces cells with structural, functional, and biochemical properties that most closely resemble those present in the fetal heart. Here we establish an in vitro engineered developmental cardiac niche to produce matured hPSC-derived cardiomyocytes (hPSC-CMs) with enhanced sarcomere development, electrophysiology, contractile function, mitochondrial capacity, and a more mature transcriptome. When this developmental cardiac niche was applied to dystrophin mutant hPSC-CMs, a robust disease phenotype emerged, which was not observed in non-matured diseased hPSC-CMs. Matured dystrophin mutant hPSC-CMs exhibited a greater propensity for arrhythmia as measured via beat rate variability, most likely due to higher resting cytosolic calcium content. Using a custom nanopatterned microelectrode array platform to screen functional output in hPSC-CMs exposed to our engineered developmental cardiac niche, we identified calcium channel blocker, nitrendipine, mitigated hPSC-CM arrhythmogenic behavior and correctly identified sildenafil as a false positive. Taken together, we demonstrate our developmental cardiac niche platform enables robust hPSC-CM maturation allowing for more accurate disease modeling and predictive drug screening.