αMHC-GFP hESCs were cultured under differentiation conditions for 8 days. (A) Day 8 hEBs were suspended as single cells and sorted for GFP expression. Propidium iodide was used to discriminate live versus dead cells. A typical sort is shown. (B) GFP⁺ cells were re-cultured under differentiation conditions for an additional 6 days, then analyzed in situ by indirect immunofluorescence for expression of cTnT and cardiac α-actinin. Cells were counterstained with DAPI to identify nuclei. Typical micrographs are shown. Expression of cTnT and cardiac α-actinin was seen throughout the culture, which demonstrated multiple foci of spontaneous contractile activity (Supplementary Videos 1 and 2). Bar, 400 µm.

αMHC-GFP hESCs (H9; 46,XX) passaged ten times after stable selection were analyzed by array comparative genomic hybridization. The log₂ ratios for all loci on the array were plotted in genome order from chromosome 1pter to Xqter. The data were normalized so that the log₂ ratio = 0 for genomic regions that are present in diploid copy number. Male reference DNA was used in the hybridization as indicated by the sex mismatch for chromosome X (arrow). There was no evidence of copy loss or gain, consistent with maintenance of euploidy.

αMHC-GFP or wild type hESCs were differentiated for 21 days, and beating GFP⁺ or wild type hEBs were manually dissected for further analysis. (A) GFP⁺ hEBs were analyzed for expression of Islet-1 (ISL-1), sarcolipin (SLN), Na⁺/K⁺ hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4), myosin light chain-2 atrial (MLC2a), MLC2v, αMHC and cTnT, relative to undifferentiated hESCs. Gene expression patterns consistent with embryonic atrium, sinoatrial (SA) node, left (LV) and right (RV) ventricle were seen. Data shown represent mean±s.e.m. for at least three hEBs with similar expression profiles for these seven genes. (B) A typical GFP⁺ hEB cultured on a gelatin-coated microelectrode array for 2 days is shown. (C) Sample field potential (FP) tracings recorded at arrayed microelectrodes are shown for HL-1 cells and typical αMHC-GFP⁺ or wild type hEBs. (D) Field potentials of averaged HL-1 cultures or hEBs were analyzed, and the number of tracings with a given FP duration (FP_dur) or decay of extracellular potential (FP_rise) were plotted as a function FP_dur (left) or FP_rise (right). Compared to the homogeneous FP recordings of HL-1 cultures, both αMHC-GFP⁺ (n = 7) and wild type (n = 5) hEBs showed similarly heterogeneous, averaged FP recordings, consistent with a heterogeneous distribution of action potential durations extracellular potential decay.

hESCs from either the αMHC-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 αMHC-GFP hESCs. In contrast, co-localization of GFP with all four markers was seen in differentiated UbiC-GFP hESCs. Bar, 100 µm.

Teratomas formed from αMHC-GFP hESCs by renal capsule grafting in SCID mice were analyzed by hematoxylin and eosin staining to identify tissue structures (left), and immunohistochemistry with anti-GFP antibody (right) to identify tissues arising from hMPs. Section from typical teratoma shown (n = 7). Primitive neural epithelium (star) and glandular intestinal structures (arrowheads) can be seen surrounding striated muscle (arrows) (left), which stains positive for GFP expression (brown; right). GFP was expressed exclusively in mononucleated myofibers within the teratomas, and was not found in any other tissues (not shown). Bar, 100 µm.

αMHC-GFP hESCs were differentiated for 12 days and the number of hMPs (GFP⁺αMHC⁺) cells were scored as a percentage of total hESC number on each day. On days 1–4, before the onset of GFP expression, unsorted differentiating hESCs were analyzed by qPCR. On days 5–12, sorted GFP⁺ hMPs were analyzed by qPCR for expression of cTnT, NKX2-5, GATA4 and αMHC, relative to undifferentiated hESCs at day 1. Data shown represent mean±s.e.m. (N = 3). Reporter activation was first observed on day 5, with 15% GFP⁺ (αMHC⁺) hESCs seen by day 9 (top). Endogenous αMHC, GATA4 and NKX2-5 expression was observed with activation of the reporter on day 5, while strong expression of cTnT did not appear until day 11 (bottom).

Relative gene expression in undifferentiated αMHC-GFP hESCs, hMPs sorted from day 8 αMHC-GFP hEBs, and hMPs sorted from day 14 αMHC-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.