Figure 7. Expression profiling of differentiating hMPs. Relative gene expression in undifferentiated aMHC-GFP hESCs, hMPs sorted from day 8 aMHC-GFP hEBs, and hMPs sorted from day 14 aMHC-GFP hEBs (two samples each) was analyzed. Heat map shows log2-fold change (sample intensity/mean intensity for undifferentiated group) for the 830 genes that had statistically significant differences (false discovery rate (FDR) ,0.05) of at least 2-fold in any pairwise comparison between the three groups. doi:10.1371/journal.pone.0016004.g007 multipotent myocardial precursor in real time. This has afforded us an unprecedented opportunity to study human CM differen- tiation 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 [24]. While these lines were able to generate electrically active CMs of unspecified subtype, MLC2v is expressed later in CM differentiation than aMHC, and does not afford the same insight into early CM differentiation. Xu et al. generated stable hESC lines using a plasmid containing the aMHC promoter driving expression of the neomycin resistance gene [25]. 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 Selection of Human Myocardial Precursors with eGFP and mCherry reporters or with puromycin resistance using the aMHC promoter [26]. 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. used Isl1:cre 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) [27], 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 [6,7,8,9,10]. 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 under- standing cardiac development and developing cell-based reagents for myocardial therapy. In summary, we have identified multipotent human myocardial precursors (hMPs) using an aMHC-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. Materials and Methods hESC culture and differentiation All work with hESCs was done with the approval of the UCSF Stem Cell Research Oversight Committee. The parent H9 hESC line (WA09; WiCell) was maintained on irradiated mouse embryonic fibroblast (MEF) feeder cells (Millipore) in a medium comprised of Knockout DMEM (Invitrogen) supplemented with 20% Knockout Serum Replacement (Invitrogen), 2 mM gluta- mine, 0.1 mM nonessential amino acids, 0.1 mM b-mercaptoeth- anol and 15 ng/ml recombinant human FGF-basic (R&D Systems). Differentiation was initiated by human embryoid body (hEB) formation in suspension as previously described [28]. Briefly, colonies of hESCs were dissociated into small clusters by exposure to Collagenase IV (Sigma-Aldrich), then allowed to differentiate in a medium comprised of Knockout DMEM (Invitrogen) supplemented with 20% Defined Fetal Bovine Serum (Hyclone), 2 mM glutamine, 0.1 mM non-essential amino acids, and 0.1 mM b-mercaptoethanol. After 4–7 days in suspension, hEBs were attached to gelatin-coated 12-well culture plates and allowed to differentiate for an additional 14–21 days. For re- culture and expression profiling experiments, hEBs were dissoci- ated with TrypLE Express (Invitrogen) to generate single cell suspensions, stained with propidium iodide to distinguish between live and dead cells, and sorted on the basis of GFP expression using a FACSAria (Becton Dickinson) with standard filter sets using previously described methods [29,30]. PLoS ONE | www.plosone.org 7 January 2011 | Volume 6 | Issue 1 | e16004