Although β-myosin heavy chain (βMHC) is the predominant isoform expressed during murine embryonic and fetal development, the transition from βMHC to αMHC begins as early as day 7.5 p.c. in mouse embryos with the appearance of somites and the onset of cardiac tube formation 
. Expression of both isoforms approaches equivalence as the cardiac tube begins to contract by day 9.5 p.c., and the β/α ratio begins to reverse with αMHC becoming the predominant isoform during postnatal life 
. These observations during mouse development in vivo
parallel mouse ESC (mESC) differentiation in vitro
. αMHC is expressed by day 8 of mESC differentiation and is expressed exclusively in beating mouse EBs 
. While many studies have used the murine αMHC promoter to track mature CMs, our data suggest that because of its expression across early and mature stages of CM development even between species, the murine αMHC promoter can be used to track CM differentiation from a multipotent myocardial precursor in real time. This has afforded us an unprecedented opportunity to study human CM differentiation from undifferentiated ESCs through EB formation and ultimately embryonic CM subtypes.
Other laboratories have developed transgenic/reporter hESC lines to derive differentiated CMs. As distinct from the work described here, however, these lines have allowed either for the selection of mature CMs only, or of progenitors that give rise to non-muscle cardiac cells in addition to myocardial cells. Huber et al.
used lentiviral vectors to produce stable hESC lines in which enhanced GFP was expressed under control of the MLC2v promoter 
. While these lines were able to generate electrically active CMs of unspecified subtype, MLC2v is expressed later in CM differentiation than αMHC, and does not afford the same insight into early CM differentiation. Xu et al.
generated stable hESC lines using a plasmid containing the αMHC promoter driving expression of the neomycin resistance gene 
. While this afforded an effective strategy for enriching CMs for cell transplant experiments, it does not accommodate the study of developmental stages over time. Kita-Matsuo et al.
recently reported the design of a set of lentiviral vectors to generate multiple stable hESC lines with eGFP and mCherry reporters or with puromycin resistance using the αMHC promoter 
. The focus of these studies was to create tools to enhance CM production for large-scale clinical application. While these investigators demonstrated that hESC-derived CMs have gene expression profiles similar to those found in adult hearts and electrophysiological properties of embryonic CMs, analysis of CM differentiation using these lines was not reported. To track the fate of human Isl1+
cells and their progeny during hESC differentiation, Bu et al.
hESCs transfected with a pCAG-flox-DsRed reporter plasmid to achieve irreversible DsRed expression in Isl1+
cells. In clonal assays of day 8 hEBs, about half of the DsRed+
clones expressed markers of the three major cardiac lineages, cTnT (CMs), PECAM1/CD31 (endothelial cells), and smooth muscle troponin (smooth muscle cells) 
, suggesting the identification of a multipotent precursor that is not restricted to the cardiac muscle lineage.
It has long been appreciated that hESCs differentiate into a heterogeneous population of atrial, ventricular and specialized conduction CMs in culture 
. Whether this reflects a stochastic process in vitro
, and is driven by a combination of genetic programming and the extracellular milieu in vivo
, has not been established. Understanding the mechanisms that drive CM subtype specification, however, will be essential to both understanding cardiac development and developing cell-based reagents for myocardial therapy.
In summary, we have identified multipotent human myocardial precursors (hMPs) using an αMHC-GFP reporter hESC line. We have demonstrated that reporter activation is restricted to hESC-derived CMs differentiated in vitro and in vivo, and that the reporter does not interfere with hESC genomic stability. Importantly, we show that hMPs give rise to multiple CM subtypes and can be used to explore CM differentiation on the molecular level by expression profiling. These precursors will provide important insight into the pathways regulating human myocardial development, and provide a novel therapeutic approach to stem cell therapy for cardiac disease.