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  • Many non integrating reprogramming strategies have been inve

    2018-10-31

    Many non-integrating reprogramming strategies have been investigated over the past 8 years, such as minicircle plasmids, synthetic mRNA/miRNA, proteins, and small molecules. However, all these approaches are labor intensive, time consuming, and often inefficient. Recently, much attention has been focused on two simple vector systems: SeV and EV. After only one infection or transfection, dozens or even hundreds of iPSC colonies can be attained 2–3 weeks later. Currently, SeV is 10- to 100-times more efficient (Schlaeger et al., 2015), whereas EV is more affordable and does not demand onerous administrative approval. The cost of EV plasmid preparation and nucleofection reagent is ∼$10 per experiment, which is ∼90% lower than purchasing the SeV reprogramming kit. Using our improved EV plasmid combination, which may outcompete SeV in reprogramming efficiency, the primary advantage of SeV vanishes. A recent comparison of EV versus SeV shows that EV iPSCs have a higher occurrence of aneuploidies (12% versus 5%) (Schlaeger et al., 2015). However, an alternative explanation of the data is that the increased abnormalities may have nothing to do with the EV itself, but rather the use of shP53 in the Yamanaka EV system. It is most likely that suppression of p53, a guardian of ploidy (Aylon and Oren, 2011), accounts for the increased occurrence of aneuploidies. With this 4-ethylphenyl sulfate in mind, we did not include shP53 or SV40 big T protein in our system. Instead, we used an anti-apoptotic factor, BCL-XL, which has no reported link with genetic abnormalities. Accordingly, human iPSC lines generated with Yamanaka factors together with BCL-XL display a normal karyotype.
    Experimental Procedures
    Author Contributions
    Introduction Methods to mature human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) may provide considerable advantages to model inherited cardiac diseases in culture (Eschenhagen et al., 2015; Jung and Bernstein, 2014; Yang et al., 2014a). Early-stage hiPSC-CMs lack discernible T tubules, display rhythmic spontaneous beating with short action potential duration and slow, diffusion-limited calcium influx (Lundy et al., 2013). In addition, the 4-ethylphenyl sulfate contractile machinery of hiPSC-CMs is typically sparse and poorly organized (Gherghiceanu et al., 2011; Kamakura et al., 2013; Lundy et al., 2013). Measuring tension remains one of the least characterized aspects of excitation-contraction coupling (ECC), due to the small size and structural immaturity of hiPSC-CMs. Thus, developing methods to measure the contractile properties of hiPSC-CMs and their subcellular organelles, myofibrils, could improve our knowledge of how early-stage cardiomyocytes function during fetal development and at the earliest stages of heart disease. In multicellular preparations, such as engineered heart tissue constructs, contractile tension of hiPSC-CMs propagates from cell to cell, and tension generation is in the order of micronewtons/section (Tulloch et al., 2011). However, low cell density, sparse cell-cell coupling, and the compliance of hydrogel- or protein-based scaffolds likely underestimates tension generation and affect contractile kinetics measurements. Single hiPSC-CM tension has been estimated by contraction stress assays (Ribeiro et al., 2015) or atomic force microscopy (Liu et al., 2012; Sun et al., 2012) but at dissimilar post-differentiation times. Moreover, two-point force assays detect twitch properties along a single axis and do not include lateral stresses, including friction due to the culture surface. Individual cardiomyocytes cultured on micropost arrays (Rodriguez et al., 2011, 2014; Yang et al., 2014b) can obviate this gap by measuring the tension, velocity, and power of contraction for subcellular bundles of myofibrils at each adhesion point. However, culturing hiPSC-CMs on micropost arrays limits myofibril elongation and bundling, cell morphology maintains a high circularity index, and the resultant tension is the sum of the contraction of unaligned myofibrils developed in multiple directions.