Precise editing of human genomes in pluripotent stem cells by homology-driven repair of targeted nuclease-induced cleavage has been hindered by the difficulty of isolating rare clones. We developed an efficient method to capture rare mutational events, enabling isolation of mutant lines with single-base substitutions without antibiotic selection. This method facilitates efficient induction or reversion of mutations associated with human disease in isogenic human induced pluripotent stem cells.

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Tissue engineering approaches have the potential to increase the physiologic relevance of human iPS-derived cells, such as cardiomyocytes (iPS-CM). However, forming Engineered Heart Muscle (EHM) typically requires >1 million cells per tissue. Existing miniaturization strategies involve complex approaches not amenable to mass production, limiting the ability to use EHM for iPS-based disease modeling and drug screening. Micro-scale cardiospheres are easily produced, but do not facilitate assembly of elongated muscle or direct force measurements. Here we describe an approach that combines features of EHM and cardiospheres: Micro-Heart Muscle (μHM) arrays, in which elongated muscle fibers are formed in an easily fabricated template, with as few as 2,000 iPS-CM per individual tissue. Within μHM, iPS-CM exhibit uniaxial contractility and alignment, robust sarcomere assembly, and reduced variability and hypersensitivity in drug responsiveness, compared to monolayers with the same cellular composition. μHM mounted onto standard force measurement apparatus exhibited a robust Frank-Starling response to external stretch, and a dose-dependent inotropic response to the β-adrenergic agonist isoproterenol. Based on the ease of fabrication, the potential for mass production and the small number of cells required to form μHM, this system provides a potentially powerful tool to study cardiomyocyte maturation, disease and cardiotoxicology in vitro.

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Background: Recent advances in nanotechnology (materials with at least one dimension between 1 nm and 100 nm) have led to the use of nanomaterials in numerous medical device applications. Recently, nanomaterials have been used to create innovative biomaterials for cardiovascular applications. Specifically, carbon nanofibers (CNF) embedded in poly(lacticco-glycolic-acid) (PLGA) have been shown to promote cardiomyocyte growth compared with conventional polymer substrates, but the mechanisms involved in such events remain unknown. The aim of this study was to determine the basic mechanism of cell growth on these novel nanocomposites. Methods: CNF were added to biodegradable PLGA (50:50 PGA:PLA weight ratio) to increase the conductivity, mechanical and cytocompatibility properties of pure PLGA. For this reason, different PLGA to CNF ratios (100:0, 75:25, 50:50, 25:75, and 0:100 wt%) with different PLGA densities (0.1, 0.05, 0.025, and 0.0125 g/mL) were used, and their compatibility with cardiomyocytes was assessed. Results: Throughout all the cytocompatibility experiments, cardiomyocytes were viable and expressed important biomarkers, including cardiac troponin T, connexin-43, and alpha-sarcomeric actin (α-SCA). Adhesion and proliferation experiments indicated that a PLGA density of 0.025 g/mL with a PLGA to CNF ratio of 75:25 and 50:50 (wt%) promoted the best overall cell growth, ie, a 55% increase in cardiomyocyte density after 120 hours compared with pure PLGA and a 75% increase compared with the control at the same time point for 50:50 (wt%). The PLGA:CNF materials were conductive, and their conductivity increased as greater amounts of CNF were added to pure PLGA, from 0 S ⋅ m −1 for pure PLGA (100:0 wt%) to 5.5 × 10 −3 S ⋅ m −1 for pure CNF (0:100 wt%), as compared with natural heart tissue (ranging from 0.16 S ⋅ m −1 longitudinally to 0.005 S ⋅ m −1 transversely). Tensile tests showed that the addition of CNF increased the tensile strength to mimic that of natural heart tissue, ie, 0.15 MPa for 100% PLGA to 5.41 MPa for the 50:50 (PLGA to CNF [wt%:wt%]) ratio at 0.025 g/mL. Atomic force microscopy indicated that the addition of CNF to PLGA increased the material surface area from 10% (100:0 [PLGA to carbon nanofiber (wt%:wt%)]) to over 60% (50:50 [PLGA to carbon nanofibers (wt%:wt%)]). Lastly, the adsorption of specific proteins (fibronectin and vitronectin) showed significantly more adsorption for the 50:50 PLGA to CNF (wt%:wt%) ratio at 0.025 g/mL PLGA compared with pure PLGA, which may be why cardiomyocyte function increased on CNF-enriched composites. Conclusion:This study demonstrates that cardiomyocyte function was enhanced on 50:50 PLGA to CNF (wt%:wt%) composite ratios at 0.025 g/mL PLGA densities because they mimicked native heart tissue tensile strength/conductivity and increased the adsorption of proteins known to promote cardiomyocyte function.