In this study, human embryonic stem cell-derived cardiomyocytes were seeded onto controlled two-dimensional micropatterned features, and an improvement in sarcomere formation and cell alignment was observed in specific feature geometries. High-resolution photolithography techniques and microcontact printing were utilized to produce features of various rectangular geometries, with areas ranging from 2500μm2 to 160,000μm2. The microcontact printing method was used to pattern non-adherent poly(ethylene glycol) regions on gold coated glass slides. Matrigel and fibronectin extracellular matrix (ECM) proteins were layered onto the gold-coated glass slides, providing a controlled geometry for cell adhesion. We used small molecule-based differentiation and an antibiotic purification step to produce a pure population of immature cardiomyocytes from H9 human embryonic stem cells (hESCs). We then seeded this pure population of human cardiomyocytes onto the micropatterned features of various sizes and observed how the cardiomyocytes remodeled their myofilament structure in response to the feature geometries. Immunofluorescence was used to measure α-actinin expression, and phalloidin stains were used to detect actin presence in the patterned cells. Analysis of nuclear alignment was also used to determine how cell direction was influenced by the features. The seeded cells showed clear alignment with the features, dependent on the width rather than the overall aspect ratio of the features. It was determined that features with widths between 30μm and 80μm promoted highly aligned cardiomyocytes with a dramatic increase in sarcomere alignment relative to the long axis of the pattern. This creation of highly-aligned cell aggregates with robust sarcomere structures holds great potential in advancing cell-based pharmacological studies, and will help researchers to understand the means by which ECM geometries can affect myofilament structure and maturation in hESC-derived cardiomyocytes.
"Two/three-dimensional culture Increases organization of sarcomeric myofilaments Ou et al. 2011  Zhang et al. 2013  Increases cardiac gene expression Pal et al. 2013  Turnbull et al. 2014  Increases contractile and Ca 2+ handling protein expression Tulloch et al. 2011  Zhang et al. 2013  Promotes alignment and anisotropy Liau et al. 2011  Promotes functional maturation in general Christoforou et al. 2013  Two-dimensional alignment and groove widths between 30 and 80 μm promote alignment and improve sarcomere structures Salick et al. 2014  Mechanical stimulation Increases expression of cardiac α-actin and MYH6, and enhances expression of cardiac transcription factors Gwak et al. 2008  Improves tissue morphology and enhances active force levels Kensah et al. 2013  Increases cell alignment Tulloch et al. 2011  Schaaf et al. 2011  Thavandiran et al. 2013  Zhang et al. 2013  Increases proliferation rates Tulloch et al. 2011  Increases AP duration and upstroke velocity, but leads to a less negative MDP Schaaf et al. 2011  Increases cell size, cytoskeletal assembly and sarcomeric organization Foldes et al. 2011  Cyclic stretch improves TNNT2 and Cx43 expression, increases contraction rates and shortens calcium transients Mihic et al. 2014  Electrical stimulation Leads to better structured and organized myofilaments Lieu et al. 2013  Produces cell elongation, affects expression of a group of cardiac-related genes Chan et al. 2013  Chen et al. 2009  Improves cardiomyocyte alignment, cross-striation patterns and force development Hirt et al. 2014  Energy substrate Elicits ARVD/C phenotype of increased apoptosis, elevated lipogenesis, and impaired calcium handling in PKP2 mutants Kim et al. 2013  Galactose and fatty acids increase oxidative phosphorylation levels, reserve capacity, and maximum respiratory capacity in mitochondria Rana et al. 2012  Glucose depletion along with lactose supplementation increase cardiomyocyte purity Tohyama et al. 2013  Induction of mitochondrial biogenesis increases cardiomyocyte differentiation Prowse et al. 2012  Other Stimulating p38-MAPK increases cell size, improves sarcomere and cytoskeletal assembly Foldes et al. 2011  Heineke and Molkentin 2006  Thyroid hormone increases cardiomyocyte size, sarcomere length, contractile force and anisotropy Yang et al. 2014  Adrenergic agonists produce hypertrophy Foldes et al. 2011  IGF1 together with electrical or electromechanical stimulation improve NRVM engineered tissue function, SERCA2a and TNNT2 expression Park et al. 2014  Morgan and Black 2014  AP, action potential; ARVD/C, arrhythmogenic right ventricular dysplasia/cardiomyopathy; Cx43, connexin 43; IGF-1, insulin-like growth factor 1; MAPK, mitogenactivated protein kinase; MDP, maximal diastolic potential; NRVM, neonatal rat ventricular myocyte; TNNT2, cardiac troponin T. cardiac differentiation, as cells associated with later stages of cardiac specification (that is, mesodermal progenitors ) had an apparent loss of substrate sensitivity when compared to hESCs . "
[Show abstract][Hide abstract] ABSTRACT: Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are the most promising source of cardiomyocytes (CMs) for experimental and clinical applications, but their use is largely limited by a structurally and functionally immature phenotype that most closely resembles embryonic or fetal heart cells. The application of physical stimuli to influence hPSC-CMs through mechanical and bioelectrical transduction offers a powerful strategy for promoting more developmentally mature CMs. Here we summarize the major events associated with in vivo heart maturation and structural development. We then review the developmental state of in vitro derived hPSC-CMs, while focusing on physical (electrical and mechanical) stimuli and contributory (metabolic and hypertrophic) factors that are actively involved in structural and functional adaptations of hPSC-CMs. Finally, we highlight areas for possible future investigation that should provide a better understanding of how physical stimuli may promote in vitro development and lead to mechanistic insights. Advances in the use of physical stimuli to promote developmental maturation will be required to overcome current limitations and significantly advance research of hPSC-CMs for cardiac disease modeling, in vitro drug screening, cardiotoxicity analysis and therapeutic applications.
[Show abstract][Hide abstract] ABSTRACT: The relatively recent development of microfluidic systems with wide-ranging capabilities for generating realistic 2D or 3D systems with single or multiple cell types has given rise to an extensive collection of platform technologies useful in muscle tissue engineering. These new systems are aimed at (i) gaining fundamental understanding of muscle function, (ii) creating functional muscle constructs in vitro, and (iii) applying these constructs to a variety of applications. Use of microfluidics to control the various stimuli that promote differentiation of multipotent cells into cardiac or skeletal muscle is first discussed. Next, systems that incorporate muscle cells to produce either 2D sheets or 3D tissues of contractile muscle are described with an emphasis on the more recent 3D platforms. These systems are useful for fundamental studies of muscle biology and can also be incorporated into drug screening assays. Applications are discussed for muscle actuators in the context of microrobotics and in miniaturized biological pumps. Finally, an important area of recent study involves coculture with cell types that either activate muscle or facilitate its function. Limitations of current designs and the potential for improving functionality for a wider range of applications is also discussed, with a look toward using current understanding and capabilities to design systems of greater realism, complexity and functionality.
Progress in Biophysics and Molecular Biology 08/2014; 115(2-3). DOI:10.1016/j.pbiomolbio.2014.08.013 · 2.27 Impact Factor
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