title: 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 2500 μm(2) to 160,000 μm(2). 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 30 μm and 80 μm 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.
authors.affiliation: Wisconsin Institutes for Discovery, 330 N Orchard St, Madison, WI 53715, USA; Department of Engineering Physics, University of Wisconsin - Madison, 1500 Engineering Drive, Madison, WI 53706, USA; Materials Science Program, University of Wisconsin - Madison, 1509 University Ave, Madison, WI 53706, USA.
authors.forename: Max R
authors.initials: MR
authors.lastname: Salick
authors.affiliation: Wisconsin Institutes for Discovery, 330 N Orchard St, Madison, WI 53715, USA; Department of Biomedical Engineering, University of Wisconsin - Madison, 1550 Engineering Drive, Madison, WI 53706, USA.
authors.forename: Brett N
authors.initials: BN
authors.lastname: Napiwocki
authors.affiliation: Wisconsin Institutes for Discovery, 330 N Orchard St, Madison, WI 53715, USA; School of Mechanical and Power Engineering, East China University of Science and Technology, Shanghai, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Sha
authors.affiliation: Wisconsin Institutes for Discovery, 330 N Orchard St, Madison, WI 53715, USA; Department of Biomedical Engineering, University of Wisconsin - Madison, 1550 Engineering Drive, Madison, WI 53706, USA.
authors.forename: Gavin T
authors.initials: GT
authors.lastname: Knight
authors.affiliation: Department of Medicine, School of Medicine and Public Health, University of Wisconsin - Madison, 750 Highland Ave, Madison, WI 53706, USA.
authors.forename: Shahzad A
authors.initials: SA
authors.lastname: Chindhy
authors.affiliation: Wisconsin Institutes for Discovery, 330 N Orchard St, Madison, WI 53715, USA; Department of Medicine, School of Medicine and Public Health, University of Wisconsin - Madison, 750 Highland Ave, Madison, WI 53706, USA; WiCell Institute, 614 Walnut Street, Madison, WI 53726, USA; Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin - Madison, 1300 University Ave, Madison, WI 53706, USA.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Kamp
authors.affiliation: Wisconsin Institutes for Discovery, 330 N Orchard St, Madison, WI 53715, USA; Department of Biomedical Engineering, University of Wisconsin - Madison, 1550 Engineering Drive, Madison, WI 53706, USA.
authors.forename: Randolph S
authors.initials: RS
authors.lastname: Ashton
authors.affiliation: Wisconsin Institutes for Discovery, 330 N Orchard St, Madison, WI 53715, USA; Department of Engineering Physics, University of Wisconsin - Madison, 1500 Engineering Drive, Madison, WI 53706, USA; Materials Science Program, University of Wisconsin - Madison, 1509 University Ave, Madison, WI 53706, USA; Department of Biomedical Engineering, University of Wisconsin - Madison, 1550 Engineering Drive, Madison, WI 53706, USA. Electronic address: crone@engr.wisc.edu.
authors.forename: Wendy C
authors.initials: WC
authors.lastname: Crone
mesh_terms: D002448:Cell Adhesion; D002454:Cell Differentiation; D002460:Cell Line; D003094:Collagen / Q000737:chemistry; D004338:Drug Combinations; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D005353:Fibronectins / Q000737:chemistry; D006046:Gold / Q000737:chemistry; D006801:Humans; D007797:Laminin / Q000737:chemistry; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism; D011509:Proteoglycans / Q000737:chemistry; D012518:Sarcomeres / Q000378:metabolism*; D013499:Surface Properties; D023822:Tissue Engineering / Q000379:methods*; D054457:Tissue Scaffolds / Q000737:chemistry*
chemical_list: D004338:Drug Combinations; D005353:Fibronectins; D007797:Laminin; D011509:Proteoglycans; C059485:matrigel; D006046:Gold; D003094:Collagen
keywords: Cardiac tissue engineering; Cardiomyocyte; Cell morphology; Micropatterning; Stem cell; Surface modification