Micropattern width dependent sarcomere development in human ESC-derived cardiomyocytes

Biomaterials (Impact Factor: 8.56). 02/2014; 35(15). DOI: 10.1016/j.biomaterials.2014.02.001
Source: PubMed


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.

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    • "PDMS was subsequently allowed to set overnight and cut by scalpel to well plate dimensions. Microcontact printing was performed on the goldcoated glass sheets as described previously [27] [38]. "
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    • "Two/three-dimensional culture Increases organization of sarcomeric myofilaments Ou et al. 2011 [63] Zhang et al. 2013 [12] Increases cardiac gene expression Pal et al. 2013 [64] Turnbull et al. 2014 [13] Increases contractile and Ca 2+ handling protein expression Tulloch et al. 2011 [10] Zhang et al. 2013 [12] Promotes alignment and anisotropy Liau et al. 2011 [66] Promotes functional maturation in general Christoforou et al. 2013 [67] Two-dimensional alignment and groove widths between 30 and 80 μm promote alignment and improve sarcomere structures Salick et al. 2014 [92] Mechanical stimulation Increases expression of cardiac α-actin and MYH6, and enhances expression of cardiac transcription factors Gwak et al. 2008 [98] Improves tissue morphology and enhances active force levels Kensah et al. 2013 [99] Increases cell alignment Tulloch et al. 2011 [10] Schaaf et al. 2011 [11] Thavandiran et al. 2013 [101] Zhang et al. 2013 [12] Increases proliferation rates Tulloch et al. 2011 [10] Increases AP duration and upstroke velocity, but leads to a less negative MDP Schaaf et al. 2011 [11] Increases cell size, cytoskeletal assembly and sarcomeric organization Foldes et al. 2011 [116] Cyclic stretch improves TNNT2 and Cx43 expression, increases contraction rates and shortens calcium transients Mihic et al. 2014 [100] Electrical stimulation Leads to better structured and organized myofilaments Lieu et al. 2013 [15] Produces cell elongation, affects expression of a group of cardiac-related genes Chan et al. 2013 [102] Chen et al. 2009 [104] Improves cardiomyocyte alignment, cross-striation patterns and force development Hirt et al. 2014 [103] Energy substrate Elicits ARVD/C phenotype of increased apoptosis, elevated lipogenesis, and impaired calcium handling in PKP2 mutants Kim et al. 2013 [83] Galactose and fatty acids increase oxidative phosphorylation levels, reserve capacity, and maximum respiratory capacity in mitochondria Rana et al. 2012 [120] Glucose depletion along with lactose supplementation increase cardiomyocyte purity Tohyama et al. 2013 [121] Induction of mitochondrial biogenesis increases cardiomyocyte differentiation Prowse et al. 2012 [126] Other Stimulating p38-MAPK increases cell size, improves sarcomere and cytoskeletal assembly Foldes et al. 2011 [116] Heineke and Molkentin 2006 [117] Thyroid hormone increases cardiomyocyte size, sarcomere length, contractile force and anisotropy Yang et al. 2014 [18] Adrenergic agonists produce hypertrophy Foldes et al. 2011 [116] IGF1 together with electrical or electromechanical stimulation improve NRVM engineered tissue function, SERCA2a and TNNT2 expression Park et al. 2014 [119] Morgan and Black 2014 [118] 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 [97]. "
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