Micropattern width dependent sarcomere development in human ESC-derived cardiomyocytes

Abstract

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 2,500μm2 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. 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.

Signals from the surface modulate differentiation of human pluripotent stem cells through glycosaminoglycans and integrins

“…We anticipate that the simplicity of our approach using modular peptide-presenting surfaces will facilitate multifactorial screens to determine the combinatorial effects of insoluble and soluble signaling on stem cell fate. Defined surfaces that support cardiomyocyte differentiation (i.e., GBP-presenting surfaces), and the selectable H9 cTnT-GFP cell line we derived, will be useful tools study cardiogenesis (47). Many differentiation protocols rely on different stages in which the soluble factors are changed over time.…”

Quantitative volumetric Raman imaging of three dimensional cell cultures

“…Human-induced pluripotent stem cell-derived CMs were obtained as follows. Gibco Episomal hiPSC Line was induced to differentiate by biphasic modulation of Wnt signalling33. Briefly, cells were routinely grown on Matrigel coated plates in Essential 8 medium.…”

Transcriptional Landscape of Cardiomyocyte Maturation

“…Although PSC-CMs can be efficiently induced from PSCs (Kattman et al, 2011; Uosaki et al, 2011), proper maturation of PSC-CMs remains a critical hurdle for recapitulating the adult phenotype. Recent studies have suggested long-term culture, extrinsic stimuli, or culturing on micropatterned substrates may improve cell morphology and enhance CM maturation (Lundy et al, 2013; Salick et al, 2014; Yanagi et al, 2007; Yang et al, 2014). However, whether these cells truly mature for meaningful use as a model for adult-heart diseases has not been established.…”