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    • pkc pathway Our data suggest that surfaces remain active

    2018-11-14

    Our data suggest that surfaces remain active primarily over the course of 24 hr. Decreased activity after this time period may result from either inadequate amounts of ligand remaining on the surfaces or self-regulation of the Notch pathway, as most Notch-mediated processes require only a transient pulse of activity that in some cases lasts only a fraction of the pkc pathway (Ambros, 1999). We were able to surpass the effects of oriented Jagged-1 surfaces through treatment with multiple doses of Notch-signaling microparticles, demonstrating the promise of using this technology to repeatedly activate or maintain Notch signaling activation. Though useful, this technology may possess several limitations. Previous studies have demonstrated that, during neonatal rat cardiomyocyte maturation, expression levels of Notch-signaling components are actually downregulated over time (Collesi et al., 2008), serving to limit the proliferation of the maturing cardiomyocyte population. As both of our Notch-signaling platforms require expression of endogenous Notch receptors in our cells of interest in order to achieve successful Notch activation, the aging of hESC-derived cardiomyocytes in culture may limit their responsiveness to our Notch-signaling technologies. In addition, as neonatal rat cardiomyocytes mature and decrease Notch pathway activity, forced activation of the pathway results in pronounced activation of DNA damage checkpoint, eventually leading to mitotic arrest and cell apoptosis (Campa et al., 2008). These findings again highlight the important role our engineered signaling platforms can play in activating Notch signaling in a physiologically relevant manner.
    Experimental Procedures
    Acknowledgments
    Introduction In response to widespread efforts to commercialize differentiated stem cells (Brower, 1999), the U.S. Food and Drug Administration (FDA) established a set of regulations and guidelines for manufacturing and quality-control evaluations of human cellular and tissue-based products derived from stem cells (FDA, 2011). The recommendations outlined for evaluating differentiated stem cell phenotypes were developed specifically to address patient safety concerns such as tumorigenicity and immunologic incompatibility, due to the initial focus of the industry on regenerative-medicine applications (Fink, 2009). Concerns about patient safety may have slowed the commercialization of regenerative therapies (Fox, 2011), but the use of industrial stem cell-based products for in vitro research, particularly pharmaceutical screening applications (Placzek et al., 2009; Rubin, 2008; Thomson, 2007; Wobus and Löser, 2011), is a promising goal that can potentially be reached in the near term. Due to the mandate to test all drug compounds for potential adverse effects on the heart, in vitro cardiac toxicity screening is a particularly important application that has prompted the development of commercial stem cell-derived cardiac myocytes by a number of companies (Webb, 2009). In this context, the focus of quality assurance shifts from patient safety concerns to the development and adoption of measures that ensure these cells reliably mimic cardiac myocytes found in vivo. In order to develop quality assurance standards for assessing stem cell-derived myocyte differentiation, it is necessary to first establish the set of characteristics that reliably define cardiac myocyte identity. We reasoned that the most effective way to delineate these standards was to comprehensively evaluate the aspects of form and function that give rise to the contractile properties of cardiac myocytes in the healthy, postnatal heart (Sheehy et al., 2012). No standardized approach currently exists for evaluating cardiac differentiation. Basic characterization involves the use of one or more assays with stringencies ranging from the observation of spontaneous beating activity to electrophysiological recordings, and one of the most commonly used approaches is gene-expression profiling (Mummery et al., 2012). In addition to measuring the expression of cardiac biomarker genes (Bruneau, 2002; Ng et al., 2010), we also examined the organizational characteristics of the contractile myofibrils (Feinberg et al., 2012), the electrical activity that regulates myofibril contraction (Kléber and Rudy, 2004), and contractile force output directly (Alford et al., 2010). Since human ventricular myocytes are not readily available, we utilized commercially available murine embryonic stem cell (mESC)- and induced pluripotent stem cell (miPSC)-derived myocytes and compared them against ventricular myocytes freshly isolated from neonatal mice (neonate). Although humans and mice exhibit differences in cardiac physiology, our goal was to determine the utility of comparing industrially manufactured stem cell-derived myocytes and isolated cardiac myocytes that possess the desired phenotype, using a multifactorial comparison of high-level myocardial tissue architectural and functional characteristics.