Ventricular cardiomyocytes VCMs are the most extensively inj

Ventricular cardiomyocytes (VCMs) are the most extensively injured cardiac cell type in ischemic heart disease and, as a result, the leading cause of reduced cardiac function. Therefore, it is of great interest to generate a renewable source of VCMs from ESCs for cell-based therapies to treat heart failure. Myosin light chain 2, ventricular isoform (Mlc2v) is specifically expressed in the ventricular chamber and is required for ventricular chamber morphogenesis during mammalian cardiogenesis (Chen et al., 1998; Minamisawa et al., 1999), providing an ideal marker for the isolation of VCMs. By using an artificial reporter system in which enhanced green fluorescent protein (eGfp) is driven by a hybrid promoter composed of a fragment of Mlc2v promoter and the enhancer thymidylate synthase inhibitor of the cytomegalovirus (CMV), two groups have previously established transgenic murine ESC (mESC) and embryonic carcinoma cell (ECC) lines for the isolation of VCMs, respectively (Moore et al., 2004; Müller et al., 2000). In the present study, we established a stable reporter system using endogenous promoter specifically activated in VCMs. The Mlc2v-Cre mouse line, in which Cre is knocked into one of the endogenous Mlc2v loci, is a well-established strain for marking VCMs (Chen et al., 1998; Minamisawa et al., 1999). By breeding this Cre line with the conditional Cre reporter strain Rosa26-yellow fluorescent protein (Yfp) (Abou-Khalil et al., 2009), we generated ESC lines from the blastocysts of the double transgenic embryos (Mlc2v-Cre; Rosa26-Yfp) and isolated VCMs from these ESC lines by using in vitro differentiation. In this study, Cre-mediated removal of a stop sequence resulted in the expression of YFP under the control of endogenous Rosa26 promoter specifically in VCMs. We further showed that these ESC-derived VCMs displayed the capacity to form the functional syncytium, neonatal ventricular cardiomyocyte-like action potentials, and rhythmic intracellular calcium transients that are responsive to both chemical and electrical stimulation. This mESC line will allow the production of homogeneous, functional VCMs for cell-based ventricular repair and regeneration in murine heart injury models. This study will set the stage for the isolation of human VCMs using MLC2V as marker in human ESCs and induced pluripotent stem cells (iPSCs) for use in cardiac repair.
Results
Discussion MLC2v is expressed exclusively in the ventricles (Chen et al., 1998; Minamisawa et al., 1999), where it contributes to the formation of sarcomeres and increases Ca2+ sensitivity at submaximal Ca2+ concentrations (Chen et al., 1998; Olsson et al., 2004). The present study demonstrates that Mlc2v-Cre; Rosa26-Yfp mESCs can differentiate into cardiac lineages and that VCMs can be isolated based on YFP expression. Several groups have reported that transplantation of cardiomyocytes derived from ESCs improves cardiac function after myocardial injury in rodent animal models (Laflamme et al., 2007; Klug et al., 1996; van Laake et al., 2007). However, successful therapy with ESC-derived donor cells is likely to require the generation of a highly purified cardiomyocyte population (Klug et al., 1996; Zandstra et al., 2003). Application of mixed cell populations into an injured left ventricle might trigger proarrhythmic effects due to the presence of inappropriate cell types such as nodal- and atrial-like cells (Zhang et al., 2009). In addition, a preparation containing undifferentiated cells could lead to tumorigenesis/teratoma formation. Thus, the production of homogenous and functional cardiomyocytes is a prerequisite for cell-based therapies. To date, cardiomyocyte markers like alpha myosin heavy chain (α-MHC) (Kita-Matsuo et al., 2009) and α-actinin (Kolossov et al., 1998) have been used to isolate cardiomyocytes from ESCs. However, the cardiac differentiation of ESCs leads to a heterogeneous population of nodal-, atrial-, and ventricular-like derivatives (He et al., 2003; Moore et al., 2008), and these markers lack the specificity needed for distinguishing between different types of cardiomyocytes. A significant percentage of cardiac-related deaths is due to ischemic heart disease related to myocardial infarction in the ventricles (Thom et al., 2006). Therefore, it is critical to develop stem cell-based therapies for the regeneration of injured ventricular tissue.