The development of the cardiac lineage in ES cell differentiation cultures is easily detected by the appearance of areas of contracting cells that display characteristics of cardiomyocytes. Development of the cardiomyocyte lineage has been analyzed in most detail in cultures induced with serum (Hescheler et al. 1997
; Boheler et al. 2002). As observed with the hematopoietic and vascular systems, development of the cardiomyocyte lineage progresses through distinct stages that are similar to development of the lineage in vivo. An ordered pattern of expression of cardiac genes is observed in the differentiation cultures, with expression of the transcription factors gata-4 and nkx2.5 that are required for lineage development preceding the expression of genes such as atrial natriuretic protein (ANP), myosin light chain (MLC)-2v, 
-myosin heavy chain (-MHC), 
-myosin heavy chain (-MHC), and connexin 43 that are indicative of distinct maturation stages within the developing organ in vivo (Hescheler et al. 1997; Boheler et al. 2002). Maturation of the lineage in the cultures is associated with changes in cell size and shape, progressing from small, round cells to elongated cells with well-developed myofibrils and sarcomeres (Boheler et al. 2002). Electrophysiologicial measurements of cells from different times in culture suggest that the cardiomyocyte population undergoes a change from early-stage cells with pacemaker-like activity to more terminally differentiated atrial- and ventricular-like cells (Maltsev et al. 1993; Hescheler et al. 1997; Banach et al. 2003). These changes correlate with the observed changes in cellular morphology, characteristic of each cell type.
While these studies clearly demonstrate the development of the cardiomyocyte lineage from differentiating ES cells, they are carried out in heterogeneous cultures in which these cells represent a minority of the entire population (Klug et al. 1996). As there are relatively few antibodies available for the isolation of cardiac progenitors, investigators have genetically engineered ES cells to enable specific selection of cells representing different stages of development within the lineage. ES cells have been generated to express either drug-resistance or fluorescent genes under the control of promoters that drive expression at specific stages of cardiac development. In the first of these approaches, Klug et al. (1996) expressed the neomycin-resistance gene under the control of the -cardiac MHC promoter. With G418 selection at appropriate stages of development, populations highly enriched (>99%) for cardiomyocytes were isolated. When applied to large-scale cultures, this strategy enabled the generation of large numbers of cardiomyocytes (Zandstra et al. 2003). Other strategies involve expressing the green fluorescent protein (GFP) from cardiac specific promoters including Nkx2.5 (Hidaka et al. 2003), cardiac -actin (Kolossov et al. 1998), and myosin light chain-2v (Muller et al. 2000). Expression from myosin light chain-2v was designed to specifically select for ventricular cells from the ES cell differentiation cultures (Muller et al. 2000). Cells selected on this basis displayed electrophysiological properties of ventricular cardiomyocytes, indicating that the strategy was successful.
Several different studies have begun to investigate the mechanisms regulating the development of the cardiac lineage in ES cell differentiation cultures. Parisi et al. (2003) demonstrated that the EGF-CFC factor Cripto, known to be essential for cardiomyocyte development in vivo (Ding et al. 1998), plays a pivotal role in differentiation of ES cells to the cardiac lineage. Cripto–/– ES cells display a deficiency in generating cardiomyocytes in culture that could be restored by the addition of soluble Cripto to the differentiation cultures. Of interest was the observation that the factor had to be supplied within the first 48 h of differentiation, suggesting that its primary function may be on the induction of mesoderm, which, in turn, differentiates to the cardiac lineage. Notch signaling also plays a role in cardiac development from ES cells (Schroeder et al. 2003). However, in this case, inhibition of the pathway appears to be important for cardiac differentiation, as ES cells lacking the recombination signal sequence-binding protein Jk, a downstream signaling molecule of all Notch receptors, generate more cardiac cells than wild-type ES cells. Other factors, including BMP2 and FGF2 (Kawai et al. 2004) as well as nitric oxide (Kanno et al. 2004) and ascorbic acid (Takahashi et al. 2003), have been shown to promote or improve cardiomyocyte differentiation in ES cell cultures. As observed with Cripto, the effects of BMP-2 and FGF were most dramatic when the factors were added early in the differentiation cultures, again suggesting that some of the effects may be mediated at the level of mesoderm induction. A role for FGF signaling in ES-cell-derived cardiac development is further supported by the observation that gfr1–/– ES cells show a marked defect in their ability to differentiate to cardiomyocytes (Dell'Era et al. 2003). Factors produced by visceral endoderm also appear to play a role in cardiomyocyte differentiation as coculture of ES cells with a visceral endoderm-like cell line, END-2, significantly enhanced cardiac development (Mummery et al. 2002). The nature of the END-2-derived molecules responsible for cardiac development remains to be determined.
While these studies have identified several factors that regulate cardiac development from ES cells, the precise stage at which they act and their relationship to each other remain to be elucidated. To characterize the mechanisms regulating cardiac development in the ES cell differentiation cultures in more detail, it will be necessary to use approaches that enable one to monitor and isolate the earliest stages of development within this lineage.
Transplantation of ES-cell-derived cardiomyocytes
One of the most promising applications of cardiomyocyte differentiation of ES cells is to provide a source of transplantable cells for the treatment of cardiovascular disease (Kehat and Gepstein 2003; Nir et al. 2003). Klug et al. (1996) first demonstrated that ES-cell-derived cardiomyocytes selected for -cardiac MHC expression could incorporate and survive in the hearts of dystrophic mice, following direct transplantation into the organ. More recent studies have demonstrated that mouse ES-cell-derived cells can survive for up to 32 wk following transplantation into the hearts of rats with myocardial infarction (Min et al. 2002, 2003). For these studies, areas of contracting cardiac cells were dissected from heterogeneous differentiation cultures and used for transplantation. Analysis of the animals indicated improved cardiac function in those transplanted with the ES-cell-derived cells compared to controls that received cell-free media. Histological analysis demonstrated the presence of differentiated donor-derived cardiac cells. To improve the survival of the ES-cell-derived cardiomyocytes, Yang et al. (2002), engineered the expression of VEGF in the cardiomyocytes prior to transplantation into the hearts of mice that had suffered myocardial infarction. Mice that received the VEGF-expressing cells displayed enhanced neovascularization and improved cardiac function compared with animals transplanted with wild-type cells.
Although the findings from these studies are encouraging, it is unclear to what extent the improvement is due to the myocyte function of the cells rather than to indirect effects such as induced vascular development at the site of injection. Future studies need to include the injection of noncardiac cells to control for the cardiac lineage specificity of the observed improvement. An additional issue relates to the differentiation status of transplanted cells. In the studies described above, relatively mature contracting cardiomyocytes were transplanted. It is conceivable that populations consisting of immature progenitor cells may be more effective in repair of the damaged heart, as such cells should display greater proliferative potential than the contracting cells and thus generate a larger graft. Identification and characterization of early-stage cardiac progenitors in ES cell differentiation cultures are important goals for the future.
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