human ubiquitin-C promoter (position 21225 to 26 upstream from the translation start site; [31]), amplified by PCR, or a 1.7 kb EcoRI-SalI fragment from the mouse aMHC promoter (generously provided by Jeffrey Robbins [13]) was inserted into the pENTR 59-TOPO entry vector using the pENTR 59-TOPO TA Cloning Kit (Invitrogen). A cDNA encoding GFP was inserted into the pENTR/D-TOPO entry vector using the pENTR/D-TOPO Cloning Kit (Invitrogen). The promoters and reporter gene were inserted into the double recombination site of the 2K7bsd plasmid using LR Clonase Plus Enzyme Mix (Invitrogen) according to the manufacturer’s instructions. Immunocytochemistry The lineage fate of differentiated hEBs was determined by staining 14-day-old, adherent differentiating hEBs attached to cover slips in 12-well culture plates. Cover slips were fixed with 4% paraformaldehyde, then permeabilized with 50% methanol/ 50% PBS, then 100% methanol, then 50% methanol/50% PBS/ 0.1% Triton X-100, and finally PBS/0.1% Triton X-100. hEBs were incubated with blocking buffer (PBS/10% horse serum/1% BSA/0.1% Triton X-100), then with primary antibody (1–5 mg/ ml) in blocking buffer. Primary antibodies used were mouse anti- human cardiac troponin T (LabVision/Neomarkers MS-295-P1; clone 13–11), mouse anti-human cardiac a-actinin (Sigma A7732; clone EA-53), mouse anti-human alpha-fetoprotein (Sigma A8452; clone C3), mouse anti-human nestin (R&D Systems MAB1259; clone 196908) or mouse anti-human smooth muscle actin (R&D Systems MAB1420; clone 1A4). Cover slips were washed with blocking buffer, incubated with 1:500 dilution of goat anti-mouse Alexa-Fluor 594 (Invitrogen A20185), washed with PBS/1% Triton X-100, then mounted with PBS and DAPI and analyzed by confocal microscopy using a Zeiss LSM510 META system. Alternatively, 8-day-old differentiating hEBs were sorted for GFP expression and re-cultured in differentiation medium in the presence of 10 mM p160 Rho-associated coiled- coil kinase inhibitor (Calbiochem 688000; Y-27632). At 14 days, differentiating hEBs were stained in situ in culture plates as described above using the same antibodies, and analyzed by immunofluorescence microscopy using a Nikon Microphot-FXA fluorescence/phase microscope and QImaging Retiga 2000R digital camera (Diagnostic Instruments) with MetaMorph soft- ware (Molecular Devices). Teratomas All experiments involving animals were done with the approval of the UCSF Institutional Animal Care and Use Committee. To form teratomas, 56105 hESCs were mixed with an equal volume of 1 mg/ml Phaseolus vulgaris lectin (PHA-P L1668; Sigma), pelleted, and incubated in growth medium overnight at 37uC, 5% CO2 in a 0.4 mm MILLICELL (Millipore). At least 2 cell pellets were grafted under each kidney capsule of 8-week-old female CB17 SCID-Beige mice (n = 7) using published techniques [32]. Transplanted cells formed teratomas in the recipients and were analyzed 10 weeks after grafting. Teratomas were fixed in 10% buffered formalin, embedded in paraffin, and 5 mm sections were stained with purified polyclonal rabbit anti-GFP (Molecular Probes A11120) at 1:1500 and biotinylated goat anti-rabbit IgG (Vector BA-1000). Slides were developed using the VECTAS- TAIN Elite ABC kit (Vector) and counterstained with hematoxylin and eosin to identify tissue structures. Quantitative real-time PCR For analysis of transcript expression, GFP+ hESCs were sorted by FACS at indicated time points, or beating areas from Selection of Human Myocardial Precursors approximately 20 hEBs were visualized with a Leica MZ6 microscope and manually excised using an 18 g needle. The collected tissue samples were treated with 0.05% Trypsin-EDTA to generate a single cell suspension prior to RNA isolation. RNA was isolated and cDNA synthesized from ,50,000 hEB-derived cells or proliferating hESCs using the Taqman Gene Expression Cells-to-CT kit (Ambion). cDNA was quantitated using a Nanodrop ND-1000 Spectrophotometer (Nanodrop Technolo- gies, ND Software version 3.3.0). Linear pre-amplification of target sequences was accomplished using the Applied Biosystems PreAmp system. Relative expression was determined using the TaqMan Assay (Applied Biosystems) on an ABI 7300 Real-Time PCR system with the following primer pairs (ABI): GATA4 (Hs00171403_m1), NKX2-5 (Hs00231763_m1), Is˜l1Hs 010996- 87_m1;Hs00158126_m1sarcolipin (Hs00161903_m1;Hs0188846- 4_s1), HCN4 (Hs00975492_m1;Hs00175760_m1), MLC2a (Hs0- 0221909_m1), MLC2v (Hs00166405_m1;Hs01125721_m1), aM- HC (Hs00411908_m1), cTnT (Hs00165960_m1), smooth muscle actin (Hs00242273_m1), a-fetoprotein (Hs00173490_m1), nestin (Hs00707120_s1), a1 integrin (Hs00235030_m1), a2 integrin (Hs00158148_m1), a4 integrin (Hs00168433_m1), a5 integrin (Hs00233732_m1), a6 integrin (Hs01041011_m1), a7 integrin (Hs00174397_m1), av integrin (Hs00233790_m1), b1 integrin (Hs00559595_m1), b5 integrin (Hs00609896_m1), and GAPDH (4326317E). Cycle times to detection were normalized against a reference gene, GAPDH, and relative changes were calculated using ABI Version 1.4 Sequence Detection Software. Microelectrode array analysis Individual GFP-expressing and wild type hEBs demonstrating contractile activity were mechanically dissected and plated on fibronectin-coated microelectrode arrays (MEA; Multi Channel Systems, Reutlingen, Germany). Mouse atrial HL-1 cells (gener- ously provided by William Claycomb, LSU Health Sciences Center) were cultured on MEAs as control. The MEA system consisted of a 50650 mm glass substrate with an embedded 1.461.4 mm matrix of 60 titanium nitride-gold contact electrodes with interelectrode distance of 200 mm. This allowed for simultaneous recording of extracellular field potentials (FP) from all electrodes over extended periods of time. Spontaneous electrical activity was recorded at 10 kHz. The temperature was kept at 37uC. Analysis of recordings was done using MC_Rack (Multi-Channel Systems) and a customized toolbox for MATLAB. The following parameters were determined: size of the largest negative peak (FPmin), last postive peak of the cycle (FPmax), the time interval between FPmin and FPmax (FPdur), and decay of extracellular potential (time from the onset of the FP to FPmin = FPrise) [19,20]. Array comparative genomic hybridization Genomic DNA (300–500 ng) from the aMHC-GFP reporter hESC line was isolated using the Qiaeasy DNA kit for tissue (Qiagen). DNA was labeled with Cy3 and Cy5 using the BioPrime DNA labeling system (Invitrogen), hybridized to HumArray 3.2 Human chromosome arrays, and analyzed for chromosomal composition. Differentially labeled human male reference genomic DNA was run as a control. The HumArray 3.2 array contains 2,464 bacterial artificial chromosome clones spotted in triplicate and distributed uniformly across the genome. Each clone contains at least one STS and is mapped to the human genome sequence. Clones containing unique sequences near the telomeres and genes known to be significant in cancer and medical genetics are included on these arrays. PLoS ONE | www.plosone.org 9 January 2011 | Volume 6 | Issue 1 | e16004