Using Ribonucleoprotein-based CRISPR/Cas9 to Edit Single Nucleotide on Human Induced Pluripotent Stem Cells to Model Type 3 Long QT Syndrome (SCN5A±)

Human induced pluripotent stem cells (hiPSCs) have been widely used in cardiac disease modelling, drug discovery, and regenerative medicine as they can be differentiated into patient-specific cardiomyocytes. Long QT syndrome type 3 (LQT3) is one of the more malignant congenital long QT syndrome (LQTS) variants with an SCN5A gain-of-function effect on the gated sodium channel. Moreover, the predominant pathogenic variants in LQTS genes are single nucleotide substitutions (missense) and small insertion/deletions (INDEL). CRISPR/Cas9 genome editing has been utilised to create isogenic hiPSCs to control for an identical genetic background and to isolate the pathogenicity of a single nucleotide change. In this study, we described an optimized and rapid protocol to introduce a heterozygous LQT3-specific variant into healthy control hiPSCs using ribonucleoprotein (RNP) and single-stranded oligonucleotide (ssODN). Based on this protocol, we successfully screened hiPSCs carrying a heterozygous LQT3 pathogenic variant (SCN5A±) with high efficiency (6 out of 69) and confirmed no off-target effect, normal karyotype, high alkaline phosphatase activity, unaffected pluripotency, and in vitro embryonic body formation capacity within 2 weeks. In addition, we also provide protocols to robustly differentiate hiPSCs into cardiomyocytes and evaluate the electrophysiological characteristics using Multi-electrode Array. This protocol is also applicable to introduce and/or correct other disease-specific variants into hiPSCs for future pharmacological screening and gene therapeutic development. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s12015-023-10602-5.


Introduction
Congenital long QT syndrome (LQTS) is the most common primary inherited arrhythmia syndrome that can contribute to risk of sudden death and occurs in an estimated 1 per 2,000 of the general population.Fifteen autosomal dominant genes have been identified to be associated with different LQTS sub-types [1] and single nucleotide substitutions (missense).Small insertion/deletions (INDEL's) are the predominant mode of genetic variants identified in LQTS genes.LQTS type 3 (LQT3) is the most malignant sub-type of congenital LQTS with a gain-of-function (GOF) variant of the I Na channel (encoded by SCN5A) resulting in impaired Na V 1.5 inactivation [2].This results in a peaked T wave morphology on the patient's electrocardiogram after a prolonged isoelectric ST segment [3,4] (Figs. 1, 2, and 3).
The human induced pluripotent stem cells (hiPSCs) have a major advantage for personalized medicine because they can be re-programmed from the human somatic cells of a particular patient without the ethical and technical challenges of utilising embryonic stem cells (ESCs).In addition, the hiPSCs are able to expand indefinitely in vitro due to their self-renewal capacity, and then differentiate into a variety of cell types, including cardiomyocytes.HiPSCs have tremendous potential to generate research models that faithfully reflect patient pathophysiology and underlying cellular pathological mechanisms [5,6].Despite these advantages, a major challenge in the field of disease modelling using hiPSCs is discriminating between abnormal in vitro disease phenotype, and background heterogeneity [7].To overcome this hurdle, appropriate isogenic hiPSC lines are considered the most accurate platform for disease modelling studies and controlling for genetic background effect [8,9].With this goal in mind, isogenic hiPSC lines can be generated using genome engineering methods.
In the CRISPR/Cas9 system, a conserved 3 bp DNA sequence (NGG) at the 3' end of sgRNA called protospacer adjacent motif (PAM) reading in the opposite direction to the RNA-DNA hybrid is essential for identifying the target DNA sequence by Cas9.Once specific site recognition by sgRNA occurs, Cas9-induced DNA double-stranded breaks (DSB) occurs.Following the DSB in the host cell, the damage is joined by one of two major endogenous cellular repair pathways, Non-Homologous End Joining (NHEJ) and Homology Directed Repair (HDR) [21].NHEJ is the dominant pathway with higher efficiency and can be widely used for research purposes in the development of knock-out strategies to study loss-of-function (LOF) of an encoded protein [22].Despite the NHEJ pathway being the dominant repair mechanism in vivo, it is prone to introducing error in DNA repair.HDR-mediated genome editing shows extremely high accuracy and error-free repair through the presence of either a homologous chromosomal DNA or an exogenous DNA template.Therefore, HDR is the preferred and most precise method in introducing/correcting specific pathogenic variants in vitro to recapitulate/rescue the disease genotype and phenotype, but has a relatively low efficiency that remains a research challenge [23].
The most efficient and widely used non-viral delivery process in CRISPR/Cas9 system is electroporation, which can deliver RNA/DNA/proteins into the target cells rapidly [24].In addition, Cas9 protein and sgRNA can form a DNAfree format-Ribonucleoprotein (RNP) complex [25,26], which has a lower molecular weight than plasmid DNA and can evade the inhibition by cellular mRNA mechanisms [27][28][29][30].This allows the genome editing components to be transiently expressed and then quickly cleared from the cells by degradation, thus facilitating higher editing efficiency and fewer potential off-target effects in a variety of cell types [31].To achieve optimal gene therapy technology, we also need to develop more effective delivery tools as well as more effective sgRNAs [32].In addition to Cas9/gRNA, singlestranded oligodeoxynucleotides (ssODNs) must be available at the DNA repair site as a template for HDR pathway [33].
In this protocol, we used the publicly available design tool (https:// zlab.bio/ guide-design-resou rces) and selected the best candidate sgRNA that had a high on-target potential with low off-target risk for genome editing, and designed the ssODN for our specific target variant of interest.We then electroporated the RNP complex (sgRNA/Cas9) and ssODN together into hiPSCs by using Neon Transfection.We also differentiate these hiPSCs into cardiomyocytes using a very simple protocol.These protocols can also be applied for correcting disease-associated pathogenic variants for gene therapy.

Materials sgRNA Synthesis and Analysis
• PCR oligonucleotides for sgRNA synthesis and ssODN were ordered from Integrated DNA Technologies
-Click 'Export to Excel' to download the full list of potential off-target sites.-The most highly predicted top 14 off-target sites were selected for off-target analysis based on the off-target score and number of mismatch and two additional genes in voltage-gated sodium channel (SCN) gene family (SCN8A and SCN9A).

Note:
The expected band should be around 120bp size.

Note:
A white precipitate will form after IVT step, and this contains pyrophosphate and smaller amounts of RNA.However, this will not affect the following purification.
2.7 Then sgRNA was purified using gRNA Clean Up Kit.
Briefly, the volume of IVT reaction (20μl) was adjusted to 200μl with nuclease-free water (180μl), and then mixed thoroughly with 100μl of Binding buffer.One volume (300μl) of 96% ethanol was added to the mix and then transferred into GeneJET TM RNA Purification Micro Column to purify the sgRNA.The purified sgRNA was eluted in 20μl of nuclease-free water.

Note:
The purification steps should be performed at room temperature.An additional empty centrifuge step is required, as the residual ethanol (in wash buffer) may inhibit the RNA purification.

The quality of sgRNA was determined by mixing with
RNA Loading Dye (2×) Solution and heating at 70 o C for 10 min, and then running on 2% electrophoresis with a Low Range ssRNA Ladder thereafter.

Note:
The expected band should be around 100-nt.
2.9 The concentration of sgRNA was measured by using Nanodrop 2000 Spectrophotometer, and then stored at -80 o C until required.

In vitro digestion with RNP • TIMING 6 hours
We recommend performing in vitro digestion of the sgRNA before electroporation.

Note:
The 3M Sodium acetate was added to adjust the pH to allow the DNA bind to the membrane of the column.
3.4 To form the RNP complex in vitro, the reaction was assembled as following: -Nuclease-free water (20μl) -NEBuffer r3.

Note: The converting ng/μl to nM of double-stranded DNA was based on the formula below:
Concentration in nM = (Concentration in ng/μl) / (660g/mol × PCR product size in bp) × 10^6 3.6 One microliter of Proteinase K was added and mixed thoroughly and then incubated at room temperature for 10min.3.7 The fragment analysis was performed on 1.5% Agarose gel.
Note: Two small fragments can be observed after Cas9 treatment, and the sum of the size should be the same as the PCR product.
Note: It is recommended to perform at least 2 passages after cell thawing before progressing to electroporation.

Thawing of hiPSCs
A) We added 500μl of Geltrex into 50ml Knock-Out DMEM and mixed them to get the 1× Geltrex solution (1:100 diluted).

Note:
The diluted Geltrex solution can be kept in 4 o C up to 4 weeks.B) For coating 2 wells of a 6-well plate, we added 1ml of Geltrex solution into each well.C) The plate was placed at 37 o C, 5% CO 2 incubator for 1-2 hours.D) Transfer a vial of cryopreserved hiPSCs from liquid nitrogen into a 37 o C water bath and quickly thaw the cells until only a sliver of ice remains.

Note: It is important to thaw cell rapidly to minimize any damage to the cell membrane. Don't loosen the vial during the thawing in the water bath as this may increase the risk of contamination.
E) Spray the vial with 70% ethanol and then transfer to a biological safety cabinet.F) Use a 1ml filter tip to transfer the contents from the vial to a 15ml tube with 9ml of E8 media supplementary with 10μM of Y-27632 (E8Y).[36].

Note: The addition of ROCK inhibitor Y-27632 is important to improve the survival rate of PSC recovery
G) Centrifuge at 200×g for 4min and aspirate the supernatant.H) Re-suspend the cell pellet in 2ml of E8Y media and seed onto the Geltrex-coated cell culture plate.I) Move the plate in several quick, short, back-and-forth and side-to-side motions to distribute the cell aggregates.Return the plate to the incubator.J) Media should be switched to E8 media after 24 hours, and refresh every day, until at 70~80% confluency.
Note: Ideally, the cells can reach 70~80% confluency in 3~4 days and can be passaged.

Passaging of hiPSCs
A) Aspirate media from the cultured hiPSCs.B) Rinse the cells by adding 1ml of DPBS (without Ca 2+ or Mg 2+ ), gently rock the plate and aspirate the DPBS.C) Add 400μl of Gentle Cell Dissociation Reagent (GCDR) and incubate at room temperature for 6~8min until the gaps appear between the cells located on the edge of the colonies.
Note: GCRD is an enzyme-free reagent suitable for the dissociation of hiPSCs into cell aggregates for routine passaging.The dissociation should be monitored under the microscope until the optimal time is determined based on appearance.D) Aspirate the GCDR and then add 2ml of E8 media to detach the cell aggregates.

Note: Avoid pipetting cells too much as it may decrease the cell survival after passaging.
E) Transfer the cell aggregates into Geltrex-coated 6-well plate with appropriate ratio.F) Gently move the plate to distribute the cell aggregates.
Place the plate in a 37 o C, 5%CO 2 incubator.G) The next day, change media with 2ml of E8 media.H) Refresh media every other day.
I) IPSCs will be at 70~80% confluency on day 3~4, and ready for performing another passage.

Electroporation using Neon Transfection System • TIMING 4 hours
5.1 IPSCs with approximately 70% confluency were treated with Y-27632 (ROCK inhibitor) for 2 hours at a final concentration of 10μM.

Note:
The ROCK inhibitor may result in a spindleshaped cell morphology, however, this change is temporary and will reverse after ROCK inhibitor is removed.

Note: It is very important to dissociate into single cells as the cell aggregate may affect the electroporation efficiency.
Optional: CloneR (STEMCELL Technologies, #05888) also showed very high efficiency in colony survival in our study.

5.3
The synthesized sgRNA and Cas9 protein were diluted in TE Buffer to the final concentration of 10μM (800ng and 3.2μg, respectively).
Note: The volume of RNP complex should not exceed 10% of the electroporation reaction.Note: Take care when transferring the electroporation mix and avoid air bubbles in the pipettes as it may cause arcing during electroporation and thus lead to failed transfection.
Note: It is very important to ensure the metal head of the Neon Pipette is tightly connected to the ball plunger inside of the Neon Pipette Station and to the Neon Tube.

Subcloning and single cell-derived clone expansion • TIMING 12 days
6.1 Immediately after electroporation, cells were re-suspended in E8R media.6.2 Single hiPSC's were manually picked and seeded directly into Geltrex-coated 96-well plate.
6.3 Fifteen to eighteen hours later, microscopic examination was carried out and the wells with only one hiPSC inside were labelled to expansion.6.4 The culture media was changed to E8 media two days after electroporation, and refreshed every 2 days.6.5 One week later, the single cell-derived colonies can be observed in 96-well plate and then passaged onto a 12-well plate using GCDR for expansion.6.6 Four to five days later, the hiPSC colonies were dissociated with GCDR and then ~20% hiPSCs were passaged onto a new 12-well plate for sub-culturing.The remaining ~80% cells were collected for genomic DNA extraction [41].Note: This method showed similar effectiveness and lower cost compared with genomic DNA isolation using RNeasy Blood & Tissue Kit (Qiagen).G) The next day, transfer the cryovial in the liquid nitrogen tank.
Note: Long-term storage in the -80 o C freezer may also decrease the viability of the hiPSCs post thawing.
Table 1 The information of potential off-target sites and the primer design 9.3 The alkaline phosphatase activity was analysed using Alkaline Phosphatase Staining Kit II.Briefly, the day 3 hiPSCs were fixed and washed with DPBS (containing 0.05% Tween-20), then incubated with fresh prepared AP substrate solution in the dark at room temperature for 10~15min until image capture.9.4 Genomic DNA was extracted for molecular karyotyping array to detect copy number variant (CNV) and short tandem repeat (STR) analysis.

Note:
The genomic DNA extraction should include a pre-treatment with RNase A and Proteinase K to remove RNA and protein.
Optional: The STR analysis can be performed as Cell Line Authentication (CLA) in Eurofins Genomics, also electrophoresis gel of amplified PCR product on 16 loci sites using our designed primers [35].3.5×10^5 (350k) hiPSCs were seeded on Geltrexcoated 12-well plate and incubated with E8R media at 37 o C for 24 hours to reach 90% confluency.

Note:
The start seeding cell density is very critical for an efficient cardiomyocyte differentiation from hiPSCs.We recommend start cardiac differentiation with a cell density of 80~90% confluency within 24~48 hours after plating.

10.3
PSC Cardiomyocyte Differentiation Medium A was applied on differentiation day 0 and Medium B on day 2. From day 5, Cardiomyocyte Maintenance Medium (CMM) was applied and refreshed every other day.
Note: Keep all the cardiac differentiation media in 4 o C. Shedding of dead cells on Day 2 and day 5 is normal.

10.4
On differentiation day 14, spontaneously beating clusters of cardiomyocytes were manually dissected and then incubated at 37 o C with STEMdiff™ Cardiomyocyte Dissociation Medium for 10min to dissociate the cardiomyocyte aggregate into single cells.

3
Note: The dissociation can be stopped by adding the Cardiomyocyte Support Medium.

10.5
Centrifuge at 300×g for 5min.10.6 The cell pellet was re-suspended in STEMdiff™ Cardiomyocyte Support Medium (CSM) containing 1% RevitaCell Supplement for downstream work.The culture media was switched to CMM after 24 hours and refreshed every other day thereafter.

11.3
On differentiation Day 28, the hiPSC-CMs were washed with cold DPBS (without Ca 2+ or Mg 2+ ), spun down, and the RNA was isolated using RNeasy Mini Kit.Then the qRT-PCR was performed to assess the expression of cardiac genes.11.4 The cardiomyocytes in chamber slides were fixed and performed immunofluorescence staining to assess the cardiac markers.

12.1
The Axion BioSystems CytoView MEA 48 Plate was coated for 2 hours, with 50μg/ml of Fibronectin (by diluting in DPBS with MgCl 2 and CaCl 2 ).2+ and Mg 2+ ) can be added to the on-plate reservoirs to increase humidity and prevent fibronectin drying out during the incubation.

12.2
On Day 14, dissociated cardiomyocytes were seeded on Fibronectin-coated MEA plate (at a density of 1×10^4/10μl) and cultured with CSM containing 1% RevitaCell Supplement for 24 hours.Note: Timing is critical in this step.Adding a drop of cardiomyocytes on MEA plates for ~2 hours to ensure the cardiomyocyte attachment.

12.3
The culture media was switched to CMM and refreshed every other day.
Note: Avoid touching the electrodes when refreshing the media. 12.4 The field potential was recorded daily from Differentiation Day 17 (3 days after replating on MEA plate) using AxIS Navigator software (Version 2.5.1.10)with the recommended settings below.12.5 Temperature was monitored during measurement and kept at 37 o C for a stable ion channel function and to prevent temperature-dependent dysregulation of ion channels [44].
Note: On the day of media refreshment, cardiomyocytes were equilibrated for at least 1 hour at 37 o C and 5% CO 2 before recording field potential.

12.6
After recording, the primary recorded AxIS Raw data was analysed in CiPA Analysis Tool (Version 3.1.8)for scientific analysis.Only one electrode, which showed a significant positive or negative T-wave and reflected the signal of most electrodes in each well, was selected as the Golden Channel from each well to detect the field potential duration (FPD) [45].The corrected FPD (FPDc) was calculated using Fredericia's correction formula [46].

Anticipated Result
Immediately after electroporation, we picked a total of 192 single iPSCs and seeded into two 96-well plates.On the second day, we identified 91 wells with single cell survival (cell survival rate as 47.40%).One week later, colonies were observed in 69 wells (clone formation efficiency as 75.82%).After Sanger sequencing from the genomic DNA of these 69 colonies, 6 clones were confirmed to have successfully incorporated the desired heterozygous variant of interest introduced (HRD efficiency as 8.70%) (Fig. S1).Following this, off-target analysis of the most likely (top 14) off-target DNA sites was performed on these 6 colonies and that result showed no off-target effect detected (Fig. S2).We acknowledge the HDR efficiency could be further improved in future work, with such strategies as optimizing the Cas9/gRNA ratio, introducing synonymous variants and a silent restriction digestion site in the ssODN design.
Furthermore, the general characterization confirmed that all these 6 edited hiPSC clones showed normal karyotype, positive pluripotency markers (OCT4, SOX2, NANOG, SSEA4, TRA-1-60 and TRA-1-81) and the spontaneous differentiation capacity to three germ layers.We subsequently differentiated the hiPSC clones into a cardiac lineage and measured the electrophysiological characteristics of the resultant cardiomyocytes using a multi-electrode array system.
In summary, we provide an RNP-based CRISPR/Cas9 strategy to introduce a heterozygous variant in wild-type hiPSCs for subsequent differentiation into a cardiac lineage for further pathogenic phenotyping.This approach can also facilitate the generation of isogenic control lines from the patient-derived hiPSCs, which represents a gold standard of control when investigating a single nucleotide variable in a disease genotype-phenotype comparison.A combination of CRISPR/Cas9 techniques and stem cell modelling can be used for in vitro disease modelling, phenotype comparison, pharmacological exploration, drug development and future personalized therapy.

Fig. 1
Fig.1sgRNA and ssODN design for single nucleotide editing.(A) sgRNA design and in vitro synthesis.Firstly, the target nucleotide (pathogenic variant of interest) is located in the 3' of sgRNA and close to the Cas9 cleavage site which is normally at 3-4nt upstream (5' side) of the protospacer adjacent motif (PAM).Then a commercial kit was used to synthesize the CRISPR system consisting of a target complementary CRISPR RNA (crRNA) and an auxiliary

Fig. 2 Fig. 3
Fig. 2 Electroporation of RNP complex into hiPSCs using Neon Transfection System.(A) Prepare the electroporation reaction mix of RNP (consisting of sgRNA and Cas9 protein), cells and ssODN.Transfer 100 μl of the mixture using the Neon Pipette and connected inside of the Neon Pipette Station.(B) Select Programme '1300 V, 30 ms, 1pulse' and press 'Start' to perform the electroporation B. Programme -1300V -30ms -1 pulse 5'-TTG CGA CCA CGG CCA GGA TC-3' (-) PAM: AGG, On-target score: 57, Off-target score: 85. 1.3 A single-stranded donor oligonucleotide (ssODN) was designed based on the sgRNA to introduce the variant of interest via homology direct repair (HDR): GGT CTC AGC GAT GGT GGC TTG GTT TTG CTC CTC ATA GGC CAT TGC GAC CA GGC CAG GATC TTC ACC AGG TAG AAG GAC CCC AGG AAG ATG ACA AGC (100-nt) (*) * colour indicates the variant of interest.colour shows the PAM sequence of the chosen sgRNA.1.4 Based on the selected sgRNA (TTG CGA CCA CGG CCAGGA TC AGG), potential off-target sites were selected.

3. 3
The PCR product was purified using The QIAquick PCR Purification Kit.Briefly, the PCR reaction was mixed with 5 volumes Buffer PB (containing pH indicator I and 3M sodium acetate) and then transferred into a QIAquick Column to purify the PCR product.The purified DNA fragment was eluted in 50μl of Buffer EB (10 mM Tris-HCl; pH 8.5).

7 .
On-target PCR screening • TIMING 6 hours 7.1 Genomic DNA was isolated from the single hiPSCderived colonies (from Step 6.6).8. Collect cell pellet.Optional: Cell pellet could be frozen in -80 o C until DNA isolation.B) Re-suspend cell pellet in 200μl of DPBS (without Ca 2+ or Mg 2+ ).C) Add 20μl Proteinase K and 4μl RNase A (100mg/ml), incubate at room temperature for 2min to remove protein and RNA.D) Add 200μl Buffer AL, vortex and incubate at 56 o C for 10 min to lyse the cells, digest protein and RNA.E) Add 1μl pH indicator, and 10μl of 3M NaOAc until suspension becomes yellow.F) Add 310μl isopropanol to precipitate DNA, vortex and incubate at room temperature for 5min.G) Vortex for 30 sec, and then centrifuge at 12,000×g in 4 o C for 30min.Discard the supernatant.H) Gently add 500μl of 70% ethanol to wash the DNA pellet, spin at 12,000×g in 4 o C for 5min.Remove ethanol carefully.I) Quick spin at 12,000×g, and remove ethanol as much as possible using a 10μl tip.J) Air dry the DNA pellet at room temperature overnight.K) Resuspend DNA pellet with 20~50 μl Buffer AE and ready for PCR amplification.

7. 2 8 .hours 8 . 1 9 .month 9 . 1
The PCR was assembled and purified based on the protocols in Step 3.2 and 3.3.7.3 Purified PCR products (5ng/μl, in 15μl) were submitted to Eurofins Genomics for Sanger sequencing.Off-target Analysis • TIMING 6 The 20-nt sgRNA is designed to direct the Cas9 protein to a particular target location within a specified gene, and the Cas9 is designed to cleave DNA around 3~4bp upstream of the PAM sequence.However, the interaction between the sgRNA and genomic DNA can tolerate some potential alignment mismatch, leading to potential off-target nuclease activity elsewhere in the genome [42, 43].8.2 The genomic DNA of the screened colonies with successful editing in Step 8 was assembly using the offtarget site primers designed in Step 1.6.(Table 1) 8.3 The purified PCR products were submitted to Eurofins Genomics for Sanger sequencing.Characterization of Genome edited hiPSC clones • TIMING 1 The genome edited hiPSC clones were expanded culture in E8 media based on the protocol in Step 4. 9.2 Cryopreservation of hiPSCs When cells reached 70~80% confluency, we cryopreserved a proportion of cells for biobanking.A) Aspirate the culture media and add 1ml of E8Y media.Note: The pre-treatment of ROCK inhibitor can help increase the survival rate of hiPSCs after cryopreservation / thawing.B) Incubate at 37 o C, 5%CO 2 incubator for 1 hour.C) Detach the cells using Cell Scraper, and then transfer all the cells into a 15ml centrifuge tube.D) Centrifuge at 200×g for 4min and aspirate the supernatant.E) Re-suspend the cell pellet in 1ml of cryopreservation media (90% Knock-out Serum Replacement (KOSR) and 10% DMSO).Note: KOSR is an animal-free and xeno-free media.F) Put the cryovial in the Thermo Scientific™ Mr. Frosty™ freezing container and leave in the -80 o C freezer overnight.Note: After re-suspending cells in cryopreservation media, transfer the cryovial into -80 o C as quick as possible.The cell viability may decrease with the long-term maintenance in cryopreservation media (containing DMSO) at room temperature.

9. 5 9 . 6 10 .
The expression of pluripotency genes were assessed by quantitative real-time polymerase chain reaction (qRT-PCR), and the pluripotency proteins were evaluated by immunofluorescence staining.A) RNA was extracted using RNeasy Mini Kit.Briefly, hiP-SCs were collected and lysis in Buffer RLT, and then mixed with 1 volume of 70% ethanol.The homogenized lysate was then transferred into RNeasy spin column.The isolated RNA was eluted in DNase/RNase-free H 2 O. B) One microgram of RNA used to synthase cDNA using QuantiNova Reverse Transcription Kit.Firstly, the potential genomic DNA in the RNA samples were removed by incubated with genomic DNA Removal Mix, and then the reverse transcription (RT) reaction was performed with the RT enzyme and mix provided in the kit.C) Quantitative real-time PCR (qRT-PCR) was performed to assess the mRNA expression levels of pluripotency genes with SYBR Green Master Mix, house-keeping gene GAPDH was used as the internal control.The spontaneously differentiation capacity was assessed by in vitro embryonic body (EB) formation with cells in three germ layers endoderm, mesoderm, and ectoderm.D)The hiPSCs were dissociated and spun down in AggreWell 400 Plate and culture 24 hours to form EBs. E) The EBs were transferred to non-coated 6-well plate and cultured in suspension for 7 days in EB media:-DMEM/F-12, GlutaMAX Supplement 384ml.-KOSR100ml, Final: 20% -L-Glutamine 5ml, Final: 1% -MEM Non-Essential Amino Acid Solution (NEAA) 5ml, Final: 1% -Penicillin-Streptomycin 5ml, Final: 1% -β-mercaptoethanol 1ml, Final: 0.2% C) Individual EBs were picked up and seeded in Geltrexcoated chamber slides and cultured for spontaneous differentiation for 4 weeks.D) On day 28, the cells were fixed and performed immunofluorescence staining with three germ layer markers: SRY-Box Transcription Factor 17 (SOX17) for endoderm, alpha-actin smooth muscle (α-SMA) for mesoderm, and β3-tubulin (TUJ1) for ectoderm.E) Images were acquired by FluoView 1000 Confocal Microscope.Cardiomyocyte Differentiation from hiPSCs • TIMING 14 days 10.1 On day -1, hiPSCs in 6-well plate were treated in E8Y for 2 hours and then dissociated into single cells using GCDR.Note: Using high quality hiPSCs (with minimal or no differentiated colonies) is very important.10.2 50k/200μl cardiomyocytes were seeded on Matrigel (1:100 (v/v) diluted in KnockOut DMEM)-coated 24-well cell culture plates for RNA extraction and 10k/150μl cardiomyocytes 8-well chamber slides for immunofluorescence staining.11.2

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'Streams -Configuration -Cardiac Real-time -Spontaneous' -Digital Filter: 0.1Hz on high pass and 2,000Hz on low pass.-The threshold for detecting cardiac beat: 200μV.-Minimum and maximum beat period at 250ms and 10s (beat rate between 6 and 240 beat per minute (BPM)).-FPD Method: Polynomial Regression (calculate the field potential duration with maximal post search duration of 1s, and the pre-and post-spike detection holdoff at 50ms and 70ms respectively).