Cardiac derivatives from ESCs consist of cardiomyocytes of a

Cardiac derivatives from ESCs consist of cardiomyocytes of atrial and ventricular phenotypes as well as pacemaker-like propyl (Wobus et al., 1995). Because of the absence of specific extracellular epitopes, no live cell sorting method exists to purify pacemaker, ventricular, or atrial myocytes without genetic tagging. As such, the inherent heterogeneity of pluripotent stem cell-derived cardiomyocytes poses a critical impediment to therapeutic/diagnostic applications. For biological pacemaker applications, contaminating ventricular/atrial myocytes in the newly derived cardiac derivatives may compromise pacemaking activity from the implanted cells. Conversely, the presence of pacemaker cells may be arrhythmogenic if ESC-derived cardiomyocytes were to replace a large mass of damaged myocardium. Although implantation of human ESC-derived cardiomyocytes in the guinea pig hearts was shown to be antiarrhythmic (Shiba et al., 2012), delivery of the same cells in nonhuman primates resulted in premature ventricular contractions and ventricular tachycardia in all experimental animals (Chong et al., 2014). As an in vitro diagnostic tool, induced pluripotent stem cell-derived cardiac myocytes have been proposed as an in vitro platform for drug screen for arrhythmic diseases such as Long QT syndrome (Itzhaki et al., 2011). However, the inherent heterogeneity of these cells, complicated by the fact that the derived cardiomyocytes are largely immature (Mummery et al., 2010; Mummery et al., 2012), may result in erroneous index of how a putative drug may react in atrial or ventricular myocardium. These concerns underscore the need for attaining subtype-specific populations of cardiomyocytes. Previous work by others has tried to address this by inducing pluripotent stem cells to cardiac pacemaker-like cells with various pharmacological agents (Kleger et al., 2010; Müller et al., 2012; Wiese et al., 2011). Here, we demonstrate that SHOX2-mediated pacemaker cell potentiation leads to heightened automaticity in the EBs and that the SHOX2-EBs provide biological pacemaker function upon transplantation into the rat myocardium in vivo. Implantation of the SHOX2-EBs was performed without microsurgical excision of nonbeating areas of the EBs, further highlighting the intensified automaticity in the SHOX2-EBs. The long-term potential of ESC-derived EBs as durable biological pacemakers is an important step toward clinical realization (Jung et al., 2014). However, we do not offer the present findings as a direct prelude to long-term biological pacing tools, but rather as insights into the progression of pacemaker cell development as they are reflected in the EB system. Because embryonic development of the SAN occurs concurrently with the looping of the linear heart tube (Christoffels et al., 2004), major signaling motifs may impart a unique signature on the developing SAN from the general myocardium. Wnt signaling is indispensable for embryonic heart development (Schneider and Mercola, 2001) and can be manipulated to enrich the cardiomyocyte population from human ESCs (Lian et al., 2012; Willems et al., 2011). Since canonical Wnt signaling is necessary for maintaining mesenchymal precursors that later form the sinus horn (Norden et al., 2011), we looked for differential expression of Wnt ligands and regulators in SHOX2-EBs. On day 6+4 expression of noncanonical Wnt, ligands are mostly suppressed, while that of Wnt inhibitors are higher in SHOX2-EBs compared with control (Figure S5A), but the expression pattern reverses later at D6+14 (Figures S5B and S5C). Inhibition of endogenous Wnt generally potentiates cardiac differentiation of ESCs (Kattman et al., 2011; Paige et al., 2010). In contrast, the expression of classical Wnt inhibitors such as Dkk1, Sfrp4, and Wif1 is significantly downregulated in SHOX2-EBs at D6+4 (Figure S5C). The data point to antagonistic effects of Wnt signaling on cardiac myocyte subtype specification, manipulation of which may steer the ESCs to differentiate into a specific cardiac cell type.