hESCs and culture conditions
Low passage hESCs (H9, WiCell) were used. Similar results were obtained using HUES13 (Harvard) and hIPSCs derived in our laboratory. Undifferentiated hESCs were cultured as described 
with slight modification. Briefly, cells were cultured in Knockout Dulbecco's modified Eagle's medium (KODMEM, Invitrogen, 10829-018) supplemented with 1 mM L-glutamine with 20% Knockout Serum Replacement medium (KOSR, Invitrogen), 1 mM sodium pyruvate, 0.1 mM nonessential amino acids (NEAA, Invitrogen), 50 U/ml penicillin, 50 µg/ml streptomycin (Invitrogen), 0.1 mM beta-mercaptoethanol (Invitrogen) and 8 ng/ml basic fibroblast growth factor (bFGF, Sigma catalogue F0291-25UG). hESCs were grown on Matrigel (growth factor-reduced, BD Bioscience)-coated 6-well plates (Corning, Inc. catalogue 3506) on a feeder layer of primary MEFs from E13.5 CD-1 mice isolated as described 
. Passage 3 to 4 MEFs were gamma-irradiated with 3,000 rads (30 Grays) and plated at 104
cells per cm2
. All hESC lines were passaged following enzymatic digestion with either collagenase IV (Invitrogen, 17104-019) approximately every 7 days or Accutase (Chemicon) approximately every 10 days 
, depending on cell condition and confluency. For collagenase treatment, cells were exposed to 1 mg/ml in KODMEM, sterile filtered, at room temperature. Once the edge of colonies were about to lift from the plate, the cells were rinsed twice with DPBS (Ca2+
- and Mg2+
-free), culture medium was added and cells were mechanically dispersed into 100–500-cell clusters by trituration using a 5 ml pipette and re-plated. For Accutase treatment, cells were washed twice with DPBS and then subsequently washed with a small amount of Accutase (1× concentration, Innovative Cell Technologies) and then exposed to Accutase at room temperature. After a few minutes, when MEFs and hESC-derived fibroblasts began to lift from the plate, accutase was removed and hESCs were washed twice with DPBS (Ca2+
- and Mg2+
-free) to remove MEFs and hESC-derived fibroblasts. A third of the volume of culture medium normally used was added and the stem cells were mechanically dispersed into 10–50-cell clusters by trituration as above. Each passage was a 1
3 split ratio for collagenase IV-treated cells and 1
4 to 1
6 ratio for accutase-treated cells. Cells were routinely tested for mycoplasma (MycoAlert; Cambrex, Walkersville, MD).
Lentivirus vector design, preparation and hESC infection
The SIN18.WPRE lentiviral vector 
was modified by insertion of the promoter regions and the drug selectable or fluorescent proteins (; see Supplemental Figures S3
for individual schematics). Vectors and schematics are available at [insert web site of public source once available]
. The lineage-specific vectors included gene promoters for T/Brachyury 
[−645 bp to −1 bp relative to ATG (includes 152 bp of 5′UTR)] and αMHC 
[−5446 bp to −4 bp relative to ATG (includes non-coding exons 1,2 and UTR of exon 3)] with the stem cell selective Rex-1 (also known as zinc finger protein 42) promoter 
(−1062 bp to −357 bp relative to ATG) to direct the selectable markers. The ubiquitously expressed human PGK promoter (−528 bp to −13 bp relative to ATG) directed expression of the H2B-fluorescent fusion proteins.
SIN18.WPRE-based lentivirus production in HEK 293T cells was as previously described 
, followed by purification and concentration by ultra-centrifugation. Briefly, three plasmids (transfer vector with expression construct, the packaging plasmid pCMVΔR8.74, and the VSV-G envelope protein expression plasmid pMD.G) were mixed in a ratio of 3
1 and 293T cells were transiently transfected using calcium phosphate method and viral supernatant from the transfected plate was collected every 24 hours in serum-free Ultraculture medium (Bio-Whitttaker #12-725F) with 1 mM L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin up to 4 days after the transfection. The pooled viral supernatant was concentrated by ultracentrifugation at 21,000 rpm for 2 hours at 4°C, passed through 0.22 or 0.4 µm filters, and aliquots were used to transfect the hESCs.
For infection, confluent hESCs, in one well of a 6-well plate, were lightly dissociated with 1 mg/ml collagenase 7 days after the last passage and rinsed twice with DPBS. The small cell clumps of approximately 100 to 200 cells were resuspended in 1 ml of culture medium and collected upon settling in 15 ml conical tube for 5 min at room temperature. 500 µl of the supernatant was exchanged with fresh 400 µl fresh media and 8 µg of polybrene. Finally 100 µl of the concentrated virus supernatant was added and mixed with the cells and incubated at 37°C for 4 to 6 hrs. The cell/virus suspension was mixed occasionally during the incubation and then plated on to one or two wells of the Matrigel-coated wells with MEF cells and cultured overnight. 1 ml of the culture media was added to the cells on the next day and the virus particles were washed out 36 hours after the infection by medium change.
G418 and Blasticidin selection of drug resistant hESC lines
Four days after virus infection, hESCs were treated with either G418 (400 µg/ml) or Blasticidin (5 µg/ml) for 36 hours, rinsed twice with DPBS to remove drugs, and cultured for two to three days with daily medium change to permit recovery. Recovered cells were then treated with the same drug a second time and allowed to recover, as before, until colonies attained sufficient size and cell density for passage.
FACS purification and cloning of hESCs
diagrams the FACS isolation and clonal expansion procedure. Plates of irradiated feeder hESCs were set up six days prior to FACS of the cells intended for clonal expansion by plating parental hESCs onto Matrigel-coated 6-well plates under regular maintenance culture conditions with MEFs as above. On the morning of the day when needed as feeders, these plates were gamma-irradiated with 3,000 rads (30 Grays), rinsed twice with DPBS, and medium exchanged with fresh H9 maintenance medium.
To generate a single-cell suspension for FACS, hESCs were dispersed with Accutase (1×) for 10 to 15 minutes at room temperature and cells were collected by centrifugation at 200 rpm for 5 minutes and the medium was exchanged to regular culturing medium and kept at room temperature until use. The dissociated hESCs (adjusted to 106 cells/ml) were stained with SytoxGreen (Invitrogen) or 7-AAD (7-amino-actinomycin D, BD Bioscience) prior to sorting on a FACSVantage™. Cell debris, cell clumps, dead cells and MEFs were gated out before sorting. Dissociated hESCs were sorted in pre-warmed 100% KOSR and then diluted with pre-warmed culture media and seeded on top of the irradiated hESC feeder plates at 10,000 to 20,000 cells/well final concentration on Matrigel-coated 6-well dishes (Corning, Inc. catalogue 3506) with 20% and 40% of KOSR, respectively. Fresh medium was added occasionally but not exchanged until day 7 post-FACS and then exchanged every day thereafter. Single colonies were passaged onto irradiated MEFs in a well of a 24-well dish on day 18 after which they were expanded onto successively larger wells with each passage () with clonal cells reaching confluence in 6-well format approximately 50 days post-FACS.
schematically illustrates the protocol for obtaining Neor, Puror (or Blar, Puror) cardiomyocytes. Undifferentiated hESC colonies were plated onto Matrigel-coated 6-well dishes (Corning Inc. Cat No. 3506) that had been seeded with 42,000 MEF cells/well and cultured until use. Prior to initiation of EB formation, the cells were treated with G418 (400 µg/ml; 1×) or Blasticidin (5 µg/ml; 1×) for 36 hours to remove residual MEFs and hESC-derived fibroblasts. Because the MEFs were removed, it was necessary to exchange media at this point (day −2) and again the next day (day −1) with 50% MEF-conditioned media and 50% hESC culturing media. Differentiation was initiated by EB formation on day 0 by treatment with 1 mg/ml collagenaseIV followed by two rinses with DPBS to remove any residual MEFs. The collagenase IV-treated colonies were dispersed by mechanical pipette trituration into cell aggregates of 500 to 800 cells. Aggregates were collected into 15 ml plastic tubes in cardiogenic medium [KODMEM supplemented with 20% fetal bovine serium (FBS; Hyclone), 1 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM NEAA, 50 U/ml penicillin, 50 µg/ml streptomycin, and 0.1 mM beta-mercaptoethanol] and allowed to settle for 5 minutes at room temperature. The supernatant containing single cells and cell debris was carefully removed and the pellet rinsed twice with medium before being re-plated on low attachment plates (Corning, Inc. Costar 3171). The medium was exchanged on day 2 and on every second day thereafter. After 6 days in suspension, EBs were transferred onto 0.1% gelatin-coated bacterial culture dishes where they attached. Cardiomyocytes generally started to appear on day 9. Cardiomyocytes were purified from αMHC-Puror hESCs by treatment with 1.8 µg/ml Puromycin for 36 hours at day 12 to14 and washed twice with DPBS.
For neuronal differentiation, EBs were prepared as above but with aggregates of approximately 50 to 100 cells in neurogenic medium [DMEM/F12 medium (Invitrogen) supplemented with 0.1 mM NEAA, 1× N-2 supplement (Invitrogen, catalogue 17502), 1× B-27 supplement (Invitrogen, catalogue 17504), 25 µg/ml bFGF (Chemicon)] in place of cardiogenic EB medium.
For endodermal differentiation, EBs were prepared as above but with aggregates of approximately 100 to 200 cells in endodermal EB medium [DMEM/F12 medium supplemented with 20% FBS (Hyclone), 0.1 mM NEAA, 0.1 mM beta-mercaptoethanol].
Total RNA was extracted using acid-guanidium-phenol-chloroform and cDNA was synthesized using the QuantiTect Reverse Transcription kit (Qiagen) and amplified products measured by Syber Green incorporation on the LightCycler (Roche). The following primers were used: hAMHC_U5619, GAAGGGCATGAGGAAGAGTGA
; hAMHC_L5901, GGTTATTCCTCGTCGTGCATC
; hBMHC_U5242, AGAACACCAGCCTCATCAACC
; hBMHC_L5639, CTGTCCTCCTCCGTCTGGTAG
; hbACTIN_U, GAGCATCCCCCAAAGTTCACA
; hbACTIN_L, GCAATGCTATCACCTCCCCTG
; hPDX-1_U, CCGCAGGAACCACGATGAGA
; hPDX-1_L, GCCACAAACAACGCCAATCC
; hAFP_U, GTCGTTTTGTCTTCTCTTCC
; hAFP_L, GCCACAAATAACAGAGGAAC
. Oct-4, Nanog, Rex-1 and hTERT primers were as in 
Cells were washed with warm PBS, fixed with ice-cold MeOH at −20°C for 7 minutes and then incubated with DPBS for 10 minutes at room temperature. Cells were blocked with 1%BSA/PBS for 1 hour and then incubated in primary antibodies for 1 hour at room temperature. After three 10-minute washes with PBS, the secondary antibody solution was incubated for a period ranging from 40 minutes to overnight at 4°C and then washed three times with PBS prior to mounting with SlowFade mounting medium with DAPI (Invitrogen). Histological sections were sectioned in OCT at 8 µm and stained as above to quantify percentage of cardiomyocytes in CSs. Cardiac Troponin-I (Alomone Labs), MAP2 (Chemicon), CD31 (eBiosciences), and appropriate AlexaFluor488 (Invitrogen), Cy3 or Cy5 (Jackson ImmunoResearch) secondary antibodies were used for immunostaining.
Microscopy and DNA content determination
For cell tracking and DNA content determination, differentiating hESCs were plated in 2 ml of appropriate differentiation medium for two days prior to recording onto 0.17-mm thick Delta T glass-bottom culture dishes (Biotechs, Butler, PA) that had been coated with 0.1% gelatin for 1 hr at room temperature. The dishes were then sealed with parafilm and mounted on the stage of an inverted Nikon microscope equipped with electronically controlled shutters, filter wheels, and a 14-bit cooled CCD camera (Orca II, Hamamatsu Corporation) controlled by MetaMorph software (Molecular Devices, USA). Time-lapse images were acquired for up to several days at a time. H2BmCherry, H2BeGFP and DAPI integrated fluorescence intensity was calculated and cell tracks were created using MetaMorph and a modified version of Particle Tracking Plugin for ImageJ 
Gene Expression Microarray Analysis
Total RNA was extracted as described 
for biological triplicates of Rex-Neor
hESCs and Rex-Neor
day 40 CSs for microarray sample preparation. Total RNA with a concentration of ~1 µg, was treated with the RiboMinus human Transcriptome Isolation kit (Invitrogen) and used as input for the GeneChip® WT cDNA Synthesis and WT Terminal Labeling kits (Affymetrix), according to manufacturers instructions by the Gladstone Institutes Genomics Core. The resulting fragmented and labeled cDNA were hybridized to individual Human Exon 1.0 ST GeneChip arrays and scanned according to manufacturers' instructions. Affymetrix CEL files from these samples were combined with CEL files for the Cythera neuronal precursor differentiation datasets (Cy-ESCs and Cy-NPs), HUES6 cell line experiment (HUES6-ESCs and HUES6-NPs) and fetal human CNS stem cells (hCNS-SCs), provided by the Gage laboratory (http://www.snl.salk.edu/˜geneyeo/stuff/papers/supplementary/ES-NP
) and 33 CEL files for 11 different adult human tissues obtained from the Affymetrix website (http://www.affymetrix.com/support/technical/sample_data/exon_array_data.affx
expression values and detection p-values were obtained for all probesets using the Affymetrix program, ExpressionConsole (http://www.affymetrix.com/products/software/specific/expression_console_software.affx
). To calculate gene expression values from the exon array data, we developed a program in python called ExpressionBuilder. Expression builder aligns probeset genomic coordinates to Ensembl genes and exons along with probeset to transcript associations from the Affymetrix probeset annotation file (HuEx-1_0-st-v2.na23.hg18.probeset.csv) to identify probesets that are most common (constitutive) to all transcripts for an Ensembl gene. Constitutive gene expression values were determined from the mean of the probeset log2 intensity values of all constitutive probesets. If no constitutive probesets are present, gene expression is calculated by the mean of all gene linked probeset intensities. To determine differential expression, fold changes and T-test p-values were calculated from the log2 expression data for differentiated cell sample arrays compared to the appropriate undifferentiated hESC baseline (Rex-Neor
H9, Cythera or HUES6 lines).
Differentially expressed genes (absolute fold>2 and p<0.05) for day 40 CS samples compared to Rex-Neor
hESCs were clustered along with differentially expressed genes (same criteria) in adult heart compared to Rex-Neor
hESCs (no filtering) using the clustering method HOPACH (hierarchical ordered partitioning and collapsing hybrid) in R 
. The resulting cluster data was visualized in the program TreeView 
. Gene Ontololgy over-representation analysis and tree filtering were performed using the freely available software GO-Elite (http://www.genmapp.org/go_elite/go_elite.html
Downloadable Gene Expression Dataset
The hESC and tissue derived gene expression data can be downloaded at http://conklinwolf.ucsf.edu/informatics/Mercola/DATASET-all-tissues_all-hESCs_all_diff-rma-exon.zip
. For 3472 Ensembl gene identifiers, mean fold change and ttest p-values are provided along with log2 expression values for all in vitro and in vivo cell/tissue derived exon arrays. This data is accompanied by gene annotations including probesets for which the values are derived, associated Affymetrix transcript clusters and HOPACH cluster data (used in ).
Intracellular recordings with sharp electrode technique
CSs were plated on coverslips coated with 0.1% gelatin and the coverslips were mounted in a chamber on the stage of an inverted microscope (Olympus IX71) and superfused with extracellular DMEM containing 1.8 mM Ca2+. All experiments were conducted at 37°C and the extracellular DMEM was continuously pre-oxygenated with 95% O2/5% CO2. Sharp glass microelectrodes are fabricated with resistances of 50–200 MΩ when filled with 3 M KCl. The spontaneously beating CSs were then impaled with the microelectrodes and electrode capacitance was nullified. The intracellular recordings of APs were obtained using an AxoPatch 200B amplifier in current clamp mode and pCLAMP-10 software (Molecular Devices). Data were sampled at 10 kHz and low pass filtered at 5 kHz. The following parameters of APs with more than 10 seconds of stable baselines were measured: AP amplitude (APA), maximum diastolic potential (MDP), maximal upstroke velocity (Vmax), AP duration at 90% of the repolarization (APD90), and the cycle-length between two spontaneous APs (RR). The APD90 is corrected by heart rates with Bazett formula (APD/square root of RR).
Single cell force measurements
Contractile forces in individual cells were measured using a method of dynamic traction force microscopy 
. Briefly, polyacrylamide gels with an elastic modulus of 4 kPa were polymerized using 4% acrylamide (BioRad), 0.2% bisacrylamide (BioRad), 5% fluorescent beads (Molecular Probes), 0.1% ammonium persulfate (Sigma) and 0.5% N,N,N′,N′-Tetramethylethylenediamine (TEMED, Biorad). These gels were bound to cover slips coated with 3-aminopropyltrimethoxysilane (Sigma) and 0.5% glutaraldehyde (Sigma). Gels were coated with 0.5 mg/ml of rat tail type I collagen (Sigma) bound through the heterobifunctional crosslinker N-Sulfosuccinimidyl-6-[4′-azido-2′-nitrophenylamino] hexanoate (sulfo-SANPAH, Pierce). Contractile CSs were manually transferred to 0.1% gelatin-coated plates for 24 hours and then dispersed into single cells by incubating in 0.25% Trypsin-EDTA (Gibco) for 30 minutes at 37°C. Individual cardiomyocytes were resuspended in serum-containing media and plated onto collagen-coated polyacrylamide gels at a density of 20,500 cells per gel and allowed to attach to substrates overnight.
Individual cells were stimulated at 0.5 Hz with 0.8 ms pulses of 50 V using a platinum electrode. Images of fluorescent beads at the gel surface were taken every 15 ms (Supplemental Movie S5
). Bead displacements were tracked using a cross-correlation-based optical flow algorithm in order to map deformations across the face of the gel 
. These displacements, along with the gel elastic modulus and Poisson Ratio were used to calculate a map of shear stress on the gel surface based on the Boussinesq solution of deformation in an infinite elastic half space 
. These stresses, or traction forces, were integrated over the projected cell area to calculate force, which is then graphed versus time. Force vectors were projected along the major axis of contraction in order to calculate the reported axial force.