sion pattern, we used a subsequence of the murine aMHC promoter consisting of nucleotides 21679 through +1 relative to the translation start site of the aMHC mRNA to construct a myocardial-specific enhanced GFP reporter in the H9 hESC line. This contains MEF1 and MEF2 binding sites, two thyroid hormone response elements, the cardiac troponin T-responsive MCAT element, and SRF binding motif [13]. We used the HIV- derived, self-inactivating lentiviral vector, 2K7bsd [14], in which the HIV gag, pol, and env genes are deleted and the HIV-1 flap sequence and woodchuck hepatitis virus post-transcriptional regulatory element are included to improve infectious titer and gene expression. To mitigate against the effects of clonal variation, we isolated stable transfectants by population selection. Presum- ably, cells in which viral integration disrupted essential genes did not survive selection. Upon differentiation of the resulting aMHC- GFP hESC line by human embryoid body (hEB) formation, GFP expression was detected solely in 14 day hEBs co-expressing cardiac troponin T (cTnT) (Fig. 1). The aMHC-GFP reporter is activated solely in hESC- derived cardiomyocytes To specifically localize cardiac versus non-cardiac proteins within differentiating aMHC-GFP+ hEBs, and confirm that GFP did not inhibit the expression of non-cardiac markers, we determined co-localization of alpha-fetoprotein, a marker of primitive endoderm, nestin, a marker of neuroectoderm, and smooth muscle actin, a marker of non-cardiac mesoderm, with GFP in the reporter line, compared to the co-localization of these proteins with ubiquitin C-driven, constitutive expression of GFP in a ubiC-GFP hESC line at day 14 of hEB differerentiation. This demonstrated that while CM-specific cTnT, but not smooth muscle actin, alpha-fetoprotein or nestin, co-localized with aMHC-driven GFP expression in the aMHC-GFP reporter line, all of these markers co-localized with ubiquitin C-driven GFP expression in the ubiC-GFP line (Fig. 1). This also confirmed that Selection of Human Myocardial Precursors the absence of smooth muscle actin, alpha-fetoprotein or nestin co- expression with aMHC-GFP was not due to interference by GFP expression. To determine the fate of selected aMHC-GFP+ hEBs, we sorted hEBs for GFP expression at day 8 of differentiation, then re- cultured aMHC-GFP+ cells under differentiation conditions for an additional 6 days (day 14 of hEB differentiation) and assessed CM- specific cTnT and cardiac a-actinin expression by in situ immunocytochemistry. This showed that cells activating the reporter on day 8 generated a lawn of cTnT+ cardiac a-actinin+ CMs in culture (Fig. 2), and that these cells demonstrate spontaneous contractile activity (Movie S1 and S2). aMHC-GFP-derived myocardial precursors give rise to muscle in vivo To establish that hESCs expressing the aMHC-GFP reporter also give rise to muscle in vivo, we evaluated undifferentiated aMHC-GFP hESCs by teratoma assay. We grafted 106 hESCs beneath the kidney capsule of 8-week-old SCID mice, and analyzed for teratoma formation after 10 weeks. The aMHC- GFP hESCs consisted of tissues arising from all three embryonic germ layers, however, indirect immunohistochemical analysis showed that GFP expression was restricted to striated muscle, specifically mononucleated myofibers characteristic of cardiac muscle (Fig. 3). Restricted GFP localization to cardiac muscle in all teratomas analyzed also suggested that hESCs expressing the aMHC-GFP reporter do not form tumors comprised of other tissues. aMHC-GFP hESCs remain genetically stable It is well-established that hESCs maintained in culture are at risk for developing aneuploidy [15,16]. Therefore, it becomes especially important to confirm genomic stability in any hESC- derived line to be used for either developmental investigation or therapy. To assess the genomic integrity of the aMHC-GFP hESC Figure 1. The aMHC-GFP reporter line is cardiac-specific. hESCs from either the aMHC-GFP or UbiC-GFP reporter lines were differentiated for 14 days by hEB formation, and analyzed by direct and indirect immunofluorescence for expression of GFP and cTnT, SMA, AFP or nestin, respectively. Cells were counterstained with DAPI to identify nuclei. Typical micrographs are shown. Co-localization of GFP solely with cardiac troponin T, but not SMA, AFP or nestin, was seen in differentiated aMHC-GFP hESCs. In contrast, co-localization of GFP with all four markers was seen in differentiated UbiC-GFP hESCs. Bar, 100 mm. doi:10.1371/journal.pone.0016004.g001 PLoS ONE | www.plosone.org 2 January 2011 | Volume 6 | Issue 1 | e16004