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Modeling mutation-specific arrhythmogenic phenotypes in isogenic human iPSC-derived cardiac tissues – Scientific Reports

Cell culture

HiPSCs, namely 409B2 (RIKENBRC #HPS0076) were maintained in StemFit AK02N medium (Ajinomoto, Cat. No. RCAK02N), on a surface pre-coated with 0.5 mg/mL laminin-511 (Nippi, Cat. No. 892021) in PBS. For passage, cells were dissociated with Accumax (Innovative Cell Technologies, Cat. No. AM105-500), incubated 10 min at 37 °C, and routinely seeded at a density of 1 × 103 cells/cm2 in StemFit AK02N medium supplemented with 10 µM ROCK inhibitor Y-27632 (Wako, Cat. No. 253-00513). After 48 h, cells were cultured without ROCK inhibitor. Cells were tested negative for mycoplasma contamination.

HiPSC gene editing

HiPSCs were targeted as previously described5. Briefly, crRNA and tracrRNA (IDT, Alt-R CRISPR-Cas9) were annealed and RNP complexes were formed by mixing 61 pmol gRNA with 61 pmol Cas9 nuclease (IDT, Alt-R S.p. Cas9 Nuclease V3) at a 1:1 gRNA:Cas9 ratio and incubating for 30 min at room temperature (RT). Finally, equal amounts of ssODN repair templates (IDT, Ultramer DNA Oligonucleotides) totaling 82 pmol were added to the preformed RNP complexes in order to generate compound heterozygous mutants. gRNA and ssODN sequences are listed in Supplementary Tables 7 and 8, respectively. 1 × 106 cells resuspended in 50 µl Opti-MEM I reduced-serum medium (Life Technologies, Cat. No. 31985-062) were added to editing reagents and electroporated in a Nepa Electroporation Cuvette 1 mm gap (Nepa Gene, Cat. No. EC-001) using the NEPA21 Electroporator (Nepa Gene) instrument (Poring pulse: 125 V voltage, 2.5 ms pulse length, 50 ms pulse gap, 2 pulses, 10% pulse decay, + orientation; Transfer pulse: 20 V voltage, 50 ms pulse length, 50 ms pulse gap, 5 pulses, 40% pulse decay, ± orientation). Electroporated cells were treated with cold shock (32 °C for 48 h) and an inhibitor cocktail composed of 2 µM NU7441 and 1 µM SCR7 for 48 h in StemFit AK02N medium supplemented with 10 µM ROCK inhibitor. Afterwards, cells were cultured in StemFit AK02N medium without ROCK inhibitor until approximately 80% confluent.

Clonal analysis

Cells were harvested 7 days after EP and 400–800 cells were plated in iMatrix511-coated 6 cm dishes. 10 days after plating, colonies approximately 1 mm in diameter were picked in 5 µl of media under the microscope and transferred to a 96-well plate in StemFit AK02N medium supplemented with 10 µM ROCK inhibitor. Depending on the growth rate of picked clones and upon average confluence across the 96-well plate, cells were then split 1:3 into 3 new 96-well plates. When reaching confluence, 2 plates were harvested, resuspended in STEM-CELLBANKER GMP grade (TAKARA BIO, Cat. No. CB047) and transferred into new 96-well plates for storage at − 80 °C and 1 plate was harvested and transferred to a 96-well PCR plate for genomic DNA extraction. For this, 10 µl QuickExtract DNA Extraction Solution (Epicenter, Cat. No. QE09050) per well was added, followed by 6 min incubation at 65 °C then 2 min at 98 °C and storage at − 30 °C.

Genotyping

For genomic DNA extraction, target sequences were PCR-amplified with KAPA HiFi HS ReadyMix (Kapa Biosystems, Cat. No. KK2602), amplicons were treated with ExoSAP-IT Express reagent (Thermo Fischer Scientific, Cat. No. 75001) for enzymatic cleanup, and Sanger sequencing was prepared with the BigDye Terminator v3.1 CS Kit (Thermo Fischer Scientific, Cat. No. 4337456). Reactions were then purified by ethanol precipitation and acquired on a 3130xl Genetic Analyzer (Applied Biosystems). Sequence alignments were analyzed with Snapgene (GSL Biotech LLC), and sequence trace files with low base calling confidence were excluded from analyses. Genotyping primers are listed in Supplementary Table 9.

Karyotyping

Representative mutant clones were seeded at a density of 5 × 104 cells in T-25 flasks, outsourced to Nihon Gene Research Laboratories (Japan), and analyzed before confluence. Each clone showed 50 spreads of 46 chromosomes and none showed banding abnormalities in 20 spreads by Geimsa staining pattern. Parent 409B2 (RIKENBRC #HPS0076) karyotypes are available at the RIKEN BioResource Research Center.

Cell differentiation

Cell differentiation was performed based on our previously reported methods21,43. HiPSCs were collected by incubating them with Versene (#15040066, 0.48 mM EDTA solution; Thermo Fisher Scientific) for 5 min at 37 °C. Versene was carefully aspirated and AK02N containing Y-27632 (10 μM) was added. Cells were detached by tapping, collected, counted and seeded onto matrigel (#354230, Growth factor reduced; 1:60 dilution; Corning)-coated wells at a density of 7.2 × 104 cells/cm2 in AK02N (0.52 mL/cm2) containing Y-27632 (10 μM) and supplemented with 4 ng/mL bFGF and cultured for 2–3 days before induction until full confluence was achieved. One day before induction (d(− 1)), medium was substituted by AK02N (0.52 mL/cm2) supplemented with 4 ng/mL bFGF and matrigel (1:60 dilution). To induce cardiac differentiation, medium was switched to RPMI1640 (#21870092, 2 mM l-glutamine, Thermo Fisher Scientific) medium with 1xB27 supplement minus the insulin (insulin-; A1895601, Thermo Fisher Scientific), further supplemented with 100 ng/mL Activin A (ActA; #338-AC, R&D systems, Minneapolis, MN) on day 0 (d0), for 24 h. The medium was then changed to RPMI1640, 2 mM l-glutamine, with 1 × B27 supplement without insulin containing 10 ng/mL human bone morphogenetic protein 4 (BMP4; #314-BP, R&D) and 10 ng/mL bFGF, and cultured the cells for 3–4.5 days with no medium change, depending on the cell type.

For MC differentiation, medium was switched to RPMI1640 (insulin-, (#A1895601) supplemented with 10% fetal bovine serum (FBS, #59901101, MOREGATE; FBS, #SFBM30-2485, EQUITHCH-BIO, INC.) and 2 mM l-glutamine on d4. The same medium was refreshed every 2–3 days until the end of differentiation. For CM differentiation, medium was switched to RPMI1640 with regular B27 supplement (#17504044, Thermo Fisher Scientific) supplemented with Wnt inhibitors XAV939 (0.25 μM) and IWP4 (0.125 μM) on d5.5 and cultured for 3.5 days. On d9, culture medium was changed to RPMI1640 with B27 supplement alone and refreshed every 2–3 days until the end of differentiation.

FACS analysis

Cardiomyocyte and mesenchymal cell identity was confirmed using FACS analysis on d15 and d21 respectively. Since differences in CM purity can affect electrophysiological results, differentiated CMs with cTnT purity below 65% were excluded from further phenotyping. Additionally, cardiac tissue sheet (CTS) phenotypes were also confirmed using FACS analysis on CTS generation. Cells and CTSs were dissociated by incubating them in Accumax (Innovative Cell Technologies) for 20 min at 37 °C and stained with the LIVE/DEAD fixable Aqua dead cell staining kit (#L34957, Thermo Fisher Scientific) to remove dead cells. MCs were stained with an anti-PDGFRβ antibody (1:100, #558821, BD Bioscience) labelled with Grn-PE and an anti-Thy1 antibody (BioLegend, San Diego, USA, 1:100) labelled with APC in EDTA at room temperature for 30 min. CMs were first fixed in 4% paraformaldehyde (PFA) for 15 min and washed twice with saponin-EDTA (0.25%, Sigma-Aldrich, St. Louis, USA), following by staining with an anti-cardiac isoform of Troponin T (cTnT) antibody (1:50, #MS-295-P, mouse monoclonal, clone 13–11, Thermo Fisher Scientific) labeled with Alexa-488 using the Zenon technology (#Z25002, Thermo Fisher Scientific) according to the manufacturer’s instructions, diluted in saponin-EDTA for 30 min at room temperature. Cells were then washed twice with EDTA (for MCs) and saponin-EDTA (for CMs), suspended in EDTA and subjected to FACS analysis (10,000 events collected per sample) using the Aria II flow cytometer (BD biosciences, Franklin Lakes, NJ).

3D cardiac tissue generation

Cardiomyocytes (d15–d21 after differentiation) were dissociated by incubation with 0.25% trypsin (Thermo Fisher Scientific), and mesenchymal cells (d15–d21 after differentiation) were dissociated by incubation with Accumax (Innovative Cell Technologies).

For CTSs consisting of CMs and MCs, the cells were mixed at a 3:1 or 1:1 ratio, and plated onto a 0.1% gelatin-coated 48-multiwell UpCell® at 6.0 × 105 cells/well with 700 μL attachment medium [AM; alpha minimum essential medium (αMEM; Thermo Fisher Scientific) supplemented with 10% FBS, 5 × 10–5 M of 2-mercaptoethanol, 50 units/mL penicillin and 50 μg/mL streptomycin] containing 50 ng/mL VEGF165 and 10 μM Y-27632. After 4 days in culture, the cells were moved to room temperature. Within 30 min, cells detached spontaneously and floated in the medium as 3D CTSs. CTSs that had holes or macroscopic irregularities were excluded from further evaluation. FACS analysis of the CM positivity rate within the CTS was made when the CTS was created from temperature-sensitive culture dishes. One of the samples was sent for FACS analysis to calculate the CM positivity rate and others were placed on the MED probe.

Measurement of extracellular field potential and drug treatment

We evaluated the phenotype of hiPSC-derived 2D cardiomyocyte and 3D arrhythmia model of cardiac tissue sheets. EFPs were measured with the MED system (Alpha MED Scientific, Osaka, Japan) using a Multi-electrode device (MED) probe with 64 planer 50 μm square microelectrodes arranged in an 8 × 8 grid at 150-μm intervals (MED-P515A).

For 2D hiPSC-CM culture, the probe was sterilized as above and coated with 2.75 μg/cm2 fibronectin (BD) before use. A total of 3 × 104 cells in 2 μL of medium was spread onto the MED probe and incubated at 37 °C. After at least 180 min, medium was added, and the hiPSC-CMs were incubated with alpha minimum essential medium (αMEM) supplemented with 10% FBS, 5 × 10–5 M 2-mercaptoethanol, 50 units/mL penicillin, 50 μg/mL streptomycin, 50 ng/mL VEGF165 and 10 μM Y-27632 for 2 days. Half of the medium was changed every 2 days. Stable spontaneous EFPs were recorded from three days to twenty-one days after the initial placement after their adhesion to the electrodes had been confirmed.

For 3D CTSs, the probe was sterilized with 70% ethanol and ultraviolet irradiation and coated with 0.1% gelatine (Sigma-Aldrich) before use. A 3D CTS was spread onto the MED probe. The medium was aspirated, and the CTS was incubated at 37 °C. The rest of the procedure was performed as described above.

We measured EFPs according to a previous report with some modifications21. Samples were equilibrated for at least 30 min in a CO2 incubator in 2 mL of fresh medium prior to the measurements. After equilibration, the MED probes were maintained at 37 °C with thermo-control systems and covered with a lid through which the gas was aerated (O2:CO2:N2 = 20%:5%:75%). EFPs from spontaneously beating samples were filtered with a 1–1000 Hz bandpass filter using the MED64 System. FPD was defined as the interval from the first peak (depolarization) to the second peak (repolarization). After recording the basal state, 2 μL dimethyl sulfoxide (DMSO; Wako) was added, and EFPs were recorded for 10 min. Then, the IKr blocker E-4031 (Wako) was added to obtain the target concentrations, and EFPs were recorded in the same manner as the recordings of DMSO treatment. For control, five DMSO concentrations (0, 0.1%, 0.2%, 0.3%, and 0.4%) were verified (Supplementary Table 10). For E-4031, six drug concentrations (0.3 nM, 3 nM, 10 nM, 30 nM,100 nM and 1000 nM) were selected to evaluate dose-dependent effects (Supplementary Table 11). At each concentration, the EFP was recorded for 10 min and the FPD values from the last 30 beats were averaged and used as the dataset for FPD and waveform analysis. The waveform of one arbitrary electrode among the 64 electrodes was selected for analysis. The selection criterion was that the depolarization and repolarization wave peaks are clearly identified according to the previously reported method46.

EFPs were processed with MED64 Mobius software (Alpha MED Scientific). Beat rate, the inter-spike interval (ms), and FPD (ms) were measured. FPD was corrected for the beating rate with Fridericia’s formula (FPDcF = FPD/[inter-spike interval/1000]1/3).

Definition of arrhythmic waveform on EFP

We defined Ventricular Tachycardia (VT)-like or Torsade de Pointes (TdP) -like waveforms as arrhythmic waveform.

Ventricular Tachycardia (VT)-like waveforms satisfied the following criteria.

Torsade de Pointes (TdP)-like waveforms satisfied the following two criteria.

  • A continuous and characteristic twisting EFP waveform with variation in polarity.

  • Continuous changes in the excitation interval corresponding to twisting changes of the EFP waveform.

Assessment of arrhythmogenicity and calculations of arrhythmogenic score

To compare arrhythmogenicity, we defined “drug test induced” as those in which E4031 up to 100 nM produced arrhythmias and “not induced” as those in which no arrhythmias occurred. We designated “spontaneously induced” as those in which arrhythmias had already appeared in the culture process before drug administration.

We defined the arrhythmogenic score as 0 points for non-induced, 1 point for drug test-induced, and 2 points for spontaneously induced, and calculated the average score for each group.

Motion Vector Prediction (MVP) analysis

We used a high-precision live cell motion imaging system (Motion Vector Prediction; Sony, Tokyo, Japan). We recorded the movies in a resolution of 2048 × 2048 pixels and adjusted the frame rates in the range from 18 to 150 images per second. Motion vectors of beating cells and CTSs were calculated using a block-matching algorithm47. From the processes of motion detection and analysis, we obtained the deformation speed as positive values. We measured the chronological fluctuation of the motion vector of 262,144 points consisting of 4 × 4 pixels. We visualized the motion amplitude by color mapping all points included in the view field and analyzed the two dimensional propagation of cellular motion.

Statistical analysis

All data analyses were performed using JMP version 14.0.0 (SAS Institute, Cary, USA). Comparisons among three or more groups were performed using One-Way ANOVA, followed by Dunnett’s test or Tukey’s test. Values are shown as the mean ± SEM. P values < 0.05 were considered significant.

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