Construction of TALENs and donor vectors
We designed two targeting donor vectors, tagged by the neomycin resistance cassette (donor 1) or EGFP (donor 2), which are connected by IRES and P2A respectively and therefore driven by the endogenous MYL2 promoter. To prevent disruption of target gene expression, the tag was inserted in the adjacent intron downstream of the last exon in the MYL2 gene. We used the internal ribosome entry site (IRES) sequence in between the neomycin coding sequences and the 5′ MYL2 homologous arm in the donor construct. We later found that, for the EGFP donor construct with IRES in between the 5′ MYL2 homologous arm and the EGFP coding sequences, the expression of EGFP was hard to detect after cardiac differentiation of the engineered hESC lines. We therefore used the P2A peptidase signal in between the MYL2 and EGFP coding sequences. The TAG stop codon of the MYL2 gene was thus removed in the EGFP donor construct. The 5′-homology arm and the 3′-homology arm of the MYL2 targeting donor vector were amplified by PCR, introducing overlapping oligonucleotides to facilitate subcloning into the vector.
Evaluation of the cutting efficiency for TALENs
The 293FT cells were obtained from Shanghai SiDanSai Biotechnology Co. Ltd. Cultured cells were transfected with TALEN plasmids. Genomic DNA was then extracted after 5 days of puromycin selection. The targeted region was amplified for sequencing and T7 Endonuclease I-based Mutation Detection (New England Biolab). Briefly, PCR products were denatured and annealed before T7 Endonuclease I digestion, and finally analyzed by electrophoresis.
MYL2 targeting in hPSCs using TALEN-based homologous recombination
The human ESC line H7 used in this study was obtained from WiCell Research Institute under a specific Material Transfer Agreement. The human iPSC line was derived previously from human skin fibroblasts in our laboratory  with informed consent approved by the Bioethics Committee of Zhongshan Hospital affiliated to Fudan University. hPSCs cultured on Matrigel (growth factor reduced; BD Biosciences) were disassociated into single cells with Accutase (Invitrogen). Three million cells were electroporated with the TALEN pairs and targeting donor vectors using the Neon Transfection System (Life Technology). Cells were seeded into a Matrigel-coated 10-cm dish containing 8 ml mTeSR1 hPSC Medium (Stem Cell Technology) for recovery at 37 °C with 5% CO2. For the selection of transfected cells, 0.25 μg/ml puromycin was used. Single clones were then picked ∼ 2 weeks later to analyze whether the neomycin resistance (donor 1) or EGFP (donor 2) cassette was successfully inserted via homologous recombination using nested PCR followed by gel electrophoresis and confirmed by DNA sequencing. Correctly targeted clones were picked into six-well plates, expanded, and transfected with plasmids expressing Cre-recombinase to excise the loxP-flanked puromycin resistance cassette.
SCID/NOD mice were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. One million undifferentiated hPSCs were suspended in 20 μl Matrigel and injected into the armpit of 8-week-old SCID/NOD mice. All rats were maintained at 24 °C, with free access to food and water. Six weeks after cell delivery, tumors were dissected and fixed with 4% paraformaldehyde in PBS for hematoxylin and eosin (H&E) staining.
Histology and immunofluorescence staining
Cells were fixed in 4% paraformaldehyde and permeabilized with 0.05% Triton-X100 followed by goat serum blocking. H7 and hiPSC colonies were stained with pluripotency marker antibodies OCT3/4 (Santa Cruz), SOX2 (Abcam), Nanog (Santa Cruz), and SSEA-4 (Abcam), whereas hPSC-derived cardiomyocytes were stained with antibodies for cTNT (Abcam), Sarcomeric α-actinin (Abcam), MYL2 (Proteintech), MYL7 (Synaptic system), or EGFP (Proteintech) for 24 h at 4 °C respectively. Cells were then incubated with Alexa Fluor 594 or 488 at 37 °C for 1 h and subsequently counterstained with DAPI. For rat hearts, heart tissues were paraffin-embedded and sectioned, followed by H&E staining. The remaining tissues were embedded with optimal cutting temperature compound (OCT; Sakura Finetek, Japan) and sectioned into sections 8 mm thick. The slides were then labeled with antibodies for Laminin (Thermo Fisher Scientific), fibronectin (Abcam), and collagen III (Abcam). Images were captured with a fluorescence microscope Leica DMi8 (Leica).
Cardiac differentiation and culture
Both H7 and hiPSCs were differentiated into the cardiomyocyte lineage following modified protocols described by Lian et al. . Seven to 9 days post differentiation, a beating cluster of cells can be observed, while robust spontaneous contraction occurs by day 10. Cultures were maintained in DMEM with 10% FBS under a 37 °C and 5% CO2 air environment.
G418 selection of MYL2-positive cardiomyocytes
At 22 days post cardiac differentiation, beating cells were digested and seeded into 12-well plates. Then 100 μg/ml G418 (InvivoGen) was used for 7–8 days for the selection of drug-resistant hPSC cardiomyocytes, with medium change every 1–2 days.
Quantitative PCR analysis
Total RNA was isolated using the Trizol reagent (Life Technologies) and 3 μg total RNA was used to synthesize cDNA using the ReverTra Ace qPCR RT Kit (FSQ-101; TOYOBO) according to the manufacturer’s instructions. Quantitative RT-RCR was performed using the SYBR® Green Realtime PCR master mix (TOYOBO) on a CFX96™ Real-Time System instrument (BIO-RAD). Each reaction was run in triplicate to minimize the variation. Gene expression values were normalized to the mean expression of the housekeeping gene GAPDH. Primer sequences are listed in Additional file 1: Table S1.
Fluorescence-activated cell sorting analysis of hPSC-derived cardiomyocytes
Dissociated cell suspension was filtered with a 40-μm cell strainer (BD Falcon) to remove cell clumps, and the cells were then fixed and permeabilized using BD Cytofix/Cytoperm™ (BD Biosciences) for 30 min at 4 °C. Next, cells were incubated with the primary antibody, including the mouse anti-human TNNT2 antibody (Thermo Scientific) or the rabbit anti-human MYL2 antibody (Proteintech), followed by appropriate FITC or PE-conjugated secondary antibody. Cells were washed twice in BD perm/wash buffer, centrifuged, and resuspended in 200 μl PBS. To define the threshold for positive fluorescence, the isotype control sample was incubated with secondary antibody only. Data were collected using FACSCalibur (BD Biosciences) and analyzed using FlowJo. A total of 10,000 gated events were counted for each marker in three independent experiments.
EGFP+ cell analysis and sorting
For the analysis of EGFP+ cardiomyocytes, dissociated and filtered MYL2EGFP/w-hPSC-derived cardiomyocytes were resuspended in PBS, and then acquired with the FACSCalibur system (BD Biosciences). GFP-positive cardiomyocytes were sorted using a BD FACSAria II flow cytometer. After the cell sorting procedure, cells were collected and plated as monolayers (~10,000 cells per coverslip) on Matrigel-coated 12-well plates in DMEM with 10% FBS. Medium was changed routinely every 2 days.
Living cells were washed with ice-cold PBS and labeled with Alexa Fluor 647-conjugated Annexin V and PI, and then incubated for 15 min in the dark as recommended. Cells were detected in 1 h using the FACSCalibur system (BD Biosciences).
Cardiac action potentials were recorded in current-clamp mode from single beating cardiomyocytes with the whole-cell patch-clamp technique, using an EPC-10 amplifier (HEKA, Lambrecht, Germany). Data were acquired using PatchMaster software (HEKA) and digitized at 1 kHz. Data analysis was performed using Igor Pro (Wavemetrics, Portland, OR, USA) and Prism (Graphpad, La Jolla, CA, USA). V-like, A-like, and N-like cardiomyocytes were identified by action potential (AP) patterns recorded in normal Tyrode’s solution containing 150 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 15 mM glucose, 1.8 mM CaCl2, 1 mM Na-pyruvate, and 15 mM HEPES (pH 7.4 with NaOH). The pipette solution contained 150 mM KCl, 5 mM NaCl, 2 mM CaCl2, 10 mM HEPES, 5 mM Mg-ATP, and 5 mM EGTA (pH 7.2 with KOH).
Neonatal rat ventricular cardiomyocyte isolation and coculture
Neonatal rat ventricular cardiomyocyte (NRVM) cultures were prepared from neonatal 1-day-old Sprague–Dawley rats. The ventricles were finely minced and dissociated with 0.075% collagenase I (Ameresco) and 0.8% Trypsin (Thermo Fisher Scientific) four times, for 10 min each. The resulting cell suspensions were passed through a cell strainer (100-μm mesh pore size; BD Biosciences) to obtain a single cell suspension, and were seeded in 12-well cell culture plates (Corning Life Sciences). For establishing cocultures, on the day of NRVM isolation the hPSC-CMs were counted and mixed with the NRVMs in a ratio of 3:1 before plating. At day 1 of culture, cells were incubated with Brdu (10 μg/ml; Sigma-Aldrich) to inhibit nonmyocyte proliferation.
Cardiomyocyte maturation culture
Dissociated hPSC-derived cardiomyocytes at 20–21 days post differentiation were plated onto Matrigel-coated six-well plates in mature medium, which consists of RPMI 1640 without glucose (Gibco), 500 μg/ml bovine serum albumin (Yeasen), 213 μg/ml l-ascorbic acid 2-phosphate (Sigma-Aldrich) supplemented with 10 mM d-galactose (Sigma-Aldrich), 4 mM l-lactic acid (Sigma-Aldrich), 1 mM sodium pyruvate (Sigma-Aldrich), 20 μg/ml insulin (Sigma-Aldrich), 1× chemically defined lipid concentrate (Sigma-Aldrich), and 200 ng/ml triiodo-l-thyronine (Sigma-Aldrich). At day 2, the medium was supplemented with 100 ng/ml G418 for another 7 days. From day 9, cells were cultured in mature medium with a medium change every other day.
Perfusion and decellularization of rat hearts
Hearts were obtained from 200–250 g adult rats and cannulated into the ascending aorta with a blunt 20-gauge needle to perform retrograde coronary perfusion. First, sterile deionized water was perfused for 30 min at 2.0 ml/min, followed by perfusion with 1% sodium dodecyl sulfate (SDS) for 3 h, and 1% triton X-100 with 0.5% EDTA (PH 8.0) for another 30 min. The hearts were then washed with deionized water and phosphate buffered saline (PBS) containing 100 U/ml penicillin (Life Technologies, USA), 100 μg/ml streptomycin (Life Technologies), and 1.25 μg/ml amphotericin B (Sigma-Aldrich) for another 2 h.
Generation of 3D ventricular heart muscles
The decellularized heart ECM was cut into pieces under sterile conditions. The ECM pieces were then put in wells of 48-well plates as a sheet. The mixture of ventricular cardiomyocytes was then seeded onto the sheet at 104 cells/mm2. The ventricular heart muscles were cultured in the mature medium and changed every day.
Electrophysiological assessment of the ventricular heart muscles
Beating ventricular heart muscles were plated on a 0.1% gelatin-coated microelectrode array (MEA) probe for 2 days with mature medium and then examined by the MEA data acquisition system MEA-2100 (Multi-channel Systems). A 60-channel voltage amplifier system was used for recording ventricular heart muscles. Data analysis was performed with SPIKE2 software (CED.UK).
Data were presented as the SEM of three independent experiments. Student’s t test was used to compare two normally distributed data sets. To compare the statistical differences of multiple groups, one-way analysis of variance (ANOVA) was used. P < 0.05 was considered statistically significant.