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    • Despite the unmet medical need to date no studies

    2018-10-31

    Despite the unmet medical need, to date, no studies have demonstrated the feasibility of isolating ventricular CMs without permanently altering their genome. Prior studies used genetic modification for isolating ventricular CMs by inserting a fluorescent reporter gene driven by the MYL2 (or MLC-2v) promoter into mouse ESCs and embryonic carcinoma cell lines (Bizy et al., 2013; Lee et al., 2012; Müller et al., 2000; Zhang et al., 2011). Such genetic modification precludes clinical use of the isolated buy Tariquidar due to concerns of tumorigenicity or adverse reactions. These ventricular CMs would not be appropriate for drug development or disease modeling due to the random and permanent changes in the genome or the use of viral vectors. Further, there are no known surface markers specific for ventricular CMs, disallowing antibody-based cell sorting with flow cytometry, which is the most common method for isolating targeted cells from differentiating PSCs. Although not surface markers, several genes are known to be specifically expressed in ventricular hearts or CMs. As a ventricular-specific transcription factor, Iroquois homeobox protein 4 (IRX4) has been reported to be exclusively expressed in the ventricular myocardium while absent from both atria and the outflow tract (Bao et al., 1999). IRX4 positively regulates ventricular-chamber-specific gene expression by activating the ventricular myosin heavy chain-1 (VMHC1) gene while suppressing the expression of atrial myosin heavy chain-1 (AMHC1) (Bruneau et al., 2000; Wang et al., 2001). As a structural protein, MYL2 (or MLC-2v), one of the essential MLC-2 isoforms that is important for the contractile function of ventricular CMs, is expressed in ventricular CMs (Marionneau et al., 2005; O’Brien et al., 1993). MYL2 expression is mostly restricted to the ventricular segment of the heart with minimal expression in the outflow track during cardiogenesis (Kubalak et al., 1994; O’Brien et al., 1993). Accordingly, we have developed a method targeting an intracellular gene to purify ventricular CMs. We used a molecular beacon (MB)-based method for isolating a pure population of ventricular CMs by targeting the mRNA of the ventricular-specific transcription factor IRX4 (Figures 1A and 1B). MBs are 20- to 30-bp oligonucleotide probes with a fluorophore and a quencher at the 5′ and 3′ ends, respectively (Figures 1A and 1B) (Heyduk and Heyduk, 2002). They are designed to form a stem-loop (hairpin) structure so that the fluorophore and quencher are within close proximity and fluorescence is quenched. Hybridization of the MBs with the target mRNA opens the hairpin structure and physically separates the fluorophore from the quencher, allowing a fluorescence signal to be emitted upon excitation (Tsourkas et al., 2002). It has been demonstrated that cellular delivery of MBs does not alter the expression level of the target genes (Rhee and Bao, 2009; Rhee et al., 2008; Santangelo et al., 2006; Tsourkas et al., 2002), and MBs can be used to isolate mESCs by directly targeting specific intracellular mRNAs such as Oct4 (Rhee and Bao, 2009). Further, we demonstrated that MBs enable the enrichment of general CMs from differentiating mouse and human PSCs (Ban et al., 2013). In the present study, we developed a sophisticated approach using MBs targeting transcription factor mRNAs, which, due to their low copy numbers compared to structural protein mRNAs, are highly challenging and were not previously attempted. By designing specific MBs targeting Irx4 mRNA, we show here that functional ventricular CMs derived from differentiating mouse ESCs could be isolated with high purity. The MB-based cell isolation method is quite versatile; a wide range of specific intracellular mRNAs could be targeted to achieve high specificity, including mRNAs encoding structural proteins and transcription factors.
    Results
    Discussion Over the past decade, there has been notable advancement in the methodologies for generating PSCs (Takahashi et al., 2007; Takahashi and Yamanaka, 2006) and producing CMs from PSCs, raising the prospects of using stem-cell-derived CMs for cardiac repair (Laflamme et al., 2007; Yang et al., 2008). However, all reported CM differentiation protocols to date can generate only heterogeneous CMs mixed with other cell populations. Although several recent studies reported non-genetic methods for isolating general CMs (Dubois et al., 2011; Hattori et al., 2010), these methods still generate heterogeneous CMs, not chamber-specific CMs. Given the major role of ventricular CMs for cardiac contractile function, it is important to develop a non-genetic method to isolate ventricular CMs from differentiating PSCs for preclinical and clinical applications.