Multiple enrichment schemes have been

Multiple enrichment schemes have been devised to separate CMs from non-CMs by exploiting cell-specific physical and biological characteristics (Huber et al., 2007; Xu, 2012). Highly purified embryonic stem cell (ESC)-derived CMs populations have been isolated from ESC lines transduced with CM-specific reporter genes (Hidaka et al., 2003; Huber et al., 2007; Anderson et al., 2007; Xu et al., 2008). Additional enrichment strategies include density gradient separation of myocytes from non-myocytes (Xu et al., 2002), isolation of cardiac bodies (Xu et al., 2006) and selection of 4-ap with a high content of mitochondria using fluorescent dyes (Hattori et al., 2010). More recently, two cell surface markers, SIRPA (Signal-Regulatory Protein Alpha) and VCAM1 (Vascular Cell Adhesion Molecule), have been shown to distinguish stem cell-derived cardiac myocytes from non-cardiac myocytes using flow cytometry (Dubois et al., 2011; Uosaki et al., 2011). Although progress has been made to direct cells towards a specific phenotype (Zhang et al., 2011), cell surface markers or alternative methodologies suitable for sorting sub-populations of CMs have not been established, and to date, purified human atrial- or ventricular-like iPSC-CM populations have not been generated. As very recently reported (Knollmann, 2013), a potential approach that can be used is to transduce cells with chamber-specific fluorescent reporter construct for subsequent purification by flow cytometry. There is abundant evidence that current iPSC-CM differentiation protocols result in CMs that resemble primitive CMs (Mummery et al., 2012; Knollmann, 2013; Lundy et al., 2013). Chamber-specific expression of cardiac contractile proteins in the adult heart is well described (Hailstones et al., 1992; Franco et al., 1998) and most cardiac sarcomeric proteins acquire their chamber-specific expression patterns relatively late during development (Lyons et al., 1990; Lyons, 1994). However, the ventricular myosin light chain-2 isoform (MLC-2v) is restricted to the ventricular segment of the heart tube at day 8 post-coitum in rodents, suggesting that its ventricular specification occurs relatively early during mammalian cardiogenesis (O\'Brien et al., 1993). In contrast, the atrial myosin light chain-2 (MLC-2a) is expressed in the presumptive ventricle prior to MLC-2v, and its ventricular expression is subsequently down-regulated (Kubalak et al., 1994). In the fetal stage, MLC-2a is primarily found in the atria while MLC-2v is essentially restricted to the ventricles, albeit low levels of both MLC-2a and MLC-2v persist in the inflow tract, the atrioventricular canal and the outflow tract (Supplementary Fig. 1) (Franco et al., 1999). This suggests that MLC-2v expression may be a robust marker for hiPSC cells committed to ventricular lineage, and here we demonstrate that a human ventricular myosin light chain-2v reporter construct can be used to identify hiPSC-CMs with an early ventricular CM phenotype.
Materials and methods
Results
Discussion Human iPSC-derived cardiac myocytes have been used for disease modeling of inherited ventricular arrhythmias such as the long QT syndrome and catecholaminergic polymorphic ventricular tachycardia (Moretti et al., 2010; Itzhaki et al., 2012). Although the hiPSC-CMs tested in those studies recapitulated many of the expected electrophysiological abnormalities of the adult disease, it is uncertain whether the tested hiPSC-CMs were atrial, ventricular or nodal-like myocytes. This poses a limitation because atrial and ventricular CMs have distinct structural and functional phenotypes. Thus, separation of these cell types is critical to gain precise mechanistic insight into molecular mechanisms of chamber specific arrhythmias. Indeed, the utility of hiPSC-derived CMs for disease modeling, drug testing and cardiac regeneration will be facilitated by development of approaches to purify lineage-specific CMs, such as the one presented here.