hiPSC (FSiPS1; obtained from the National Stem Cell Bank, KNIH, Korea) were cultured in E8 medium (Gibco, Carlsbad, CA, USA) under XF conditions or in mTeSR^TM1 (STEMCELL Technologies, Vancouver, Canada) under XC conditions at 37℃ in the presence of 5% CO₂. As a coating material for the 6-well plate, Vitronectin (VTN-N) Recombinant Human Protein (Gibco) was used in the XF culture and Matrigel^TM hESC-qualified (Corning, Bedford, MA, USA) was used in the XC culture. The culture media were replaced daily, and the cells were subcultured (at a 1：7 ratio) using a non-enzymatic cell dissociation method with Versene solution (Gibco) every 5 days.

hiPSC-CM was generated by inducing the differentiation of hiPSC into cardiac-lineage cells under XF or XC conditions (Fig. 1). For XC culture, the previously reported GiAB protocol (MM.1) (19) was used, with some modifications by adding matrix Matrigel^TM at day 0 and day 1 of differentiation. Briefly, 5×10⁵ hiPSCs/well were seeded on a Matrigel^TM-coated 12-well plate and treated with mTeSR^TM1 supplemented with the GSK3 inhibitor CHIR99021 (1 μM) for 4 days. To induce the differentiation of hiPSC into mesodermal cells and cardiac-lineage cells, we used RPMI-based differentiation media, which contain various components, such as activin A, basic fibroblast growth factor (bFGF), knock-out serum replacement (KO-SR), B-27 supplements-insulin, and BMP4, in different combinations, depending on the differentiation stage. For XF culture, according to the manufacturer’s instructions, hiPSC were differentiated into CM using the Gibco PSC Cardiomyocyte Differentiation Kit (Gibco). Briefly, human iPSCs were singularized using ACCUTASE^TM, and 1×10⁵ cells/well were seeded on a VTN-N-coated 12-well plate in E8 medium. The E8 medium was replaced daily for 4 days to allow sufficient proliferation of hiPSC, and then the following media were applied sequentially: cardiomyocyte differentiation medium A, cardiomyocyte differentiation medium B, and cardiomyocyte mainte-nance medium. Ten days after differentiation, the beating of the CM was observed. All cultures were maintained at 37℃ in the presence of 5% CO₂.

Total RNA was extracted from hiPSC-CM under XF or XC conditions at 0, 2, and 4 weeks after differentiation using the RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s recommen-dations. After the quantification of RNA, complementary DNA (cDNA) was synthesized using 1 μg of RNA and an iScriptTM cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s ins-tructions. RT-qPCR was performed using a cDNA template and the QuantiTect SYBR Green PCR Kit (QIAGEN) in a real-time PCR system (7900HT Fast Real-Time PCR System, Applied Biosystems, Waltham, MA, USA). The sequences of primers used for the target genes are listed in Supplementary Table S1. The relative expression of genes was calculated using the 2^–ΔΔCtmethod with ExpressionSuite Software Version 1.2 (Thermo Fisher Scientific, Waltham, MA, USA). The expression values were normalized against those of the 18S rRNA gene as a housekeeping gene.

hiPSC-CM were fixed in 4% paraformaldehyde (Sigma, St. Louis, MO, USA) for 10 min and permeabilized with 0.1% Triton X-100 (Sigma) in PBS for 10 min. After the cells were treated with an immunofluorescence blocking buffer (Cell Signaling Technology, Danvers, MA, USA), they were treated overnight at 4℃ with the following primary antibodies: MLC-2v (1：200, Thermo Fisher Scientific, 10906-1-AP), Troponin T (1：200, ab8295, Abcam, Cam-bridge, UK), α-SMA (1：200, Cell Signaling Technology, #19245s), and α-actinin (1：100, MERCK, A7811). All primary antibodies were diluted using an immunofluo-rescence antibody dilution buffer (Cell Signaling Technology). After washing with a 1X washing buffer (Cell Signaling Technology), the cells were incubated for 1 h at room temperature with goat anti-rabbit IgG (H+L)-Alexa Fluor 555 (1：250, Thermo Fisher Scientific, A21428) and goat anti-mouse IgG (H+L)-Alexa Fluor 488 (1：250, Thermo Fisher Scientific, A11001) as fluorescence-conjugated secondary antibodies. The nuclei were stained with DAPI (NucBlue^TM Fixed Cell Stain ReadyProbes^TM Reagent, Thermo Fisher Scientific, R37606). Images of the cells were acquired using a fluorescence microscope (Axio Observer 7; Carl Zeiss).

This assay was performed as previously described (20). In brief, to attach hiPSC-CM on the assay plate, 12-well MEA plates (Axion Biosystems, Atlanta, GA, USA) containing electrodes were coated for 30 min with 50 μg/ml fibronectin in DPBS (7 μl/well). hiPSC-CM were singularized with the STEMdiff^TM Cardiomyocyte Dissociation Kit (STEMCELL Technologies, Vancouver, Canada), and 4×10⁶ cells were seeded in plating media suitable for XC- or XF-hiPSC-CM maintenance and transferred to the MEA plate. Field potentials (FPs) were recorded for spontaneously beating hiPSC-CM. During the recording period, the MEA plate was placed at 37℃ in a sterile environment using the Maestro MEA system (Axion Biosystems). After the FP waveforms were stabilized, the signals were recorded for 5 min and the last 1 min were analyzed. Fridericia’s formula (FPDcF), where FPDcF=field potential duration (FPD)/beat period^0.33, was used to correct the dependence of the FPD on the beating rate.

According to the manufacturer’s recommendations, Neu5Gc was detected using an ELISA with an Anti-Neu5Gc antibody kit (BioLegend, San Diego, CA, USA; free of Neu5Gc). Briefly, serum (10 μl/well), conditioned medium (10 μl/well), and cell lysate (500 μg/well) samples collected under XC or XF conditions were placed on a high-binding 96-well ELISA plate (Corning). After the sodium carbonate-bicarbonate immobilization buffer (50 mM, pH 9.5, Invitrogen, Carlsbad, CA, USA) was added to each well, the samples were incubated for 16∼18 h at 4℃. The wells were blocked for 1 h at room temperature with the Neu5Gc Assay Blocking Solution and incubated for 2 h with a purified anti-Neu5Gc antibody (primary antibody) and chicken IgY isotype control diluted at a ratio of 1：5,000 in PBS Tween^TM 20 buffer (PBST) (Pierce Biotechnology, Rockford, IL, USA). The plates were washed three times with PBST and subsequently incubated for 1 h at room temperature with goat anti-chicken IgY H&L (HRP) (Abcam) diluted at a 1：5,000 ratio in PBS. After washing three times with PBST, the wells were developed with a substrate reagent pack and the stop solution 2N sulfuric acid (R&D Systems, Minneapolis, MN, USA). Absorbance was measured at 490 nm using the Spectra-Max 190 with SoftMax Pro 6.3 software (Molecular Devices).

hiPSC-CM lysates were prepared at 0, 2, 4, and 6 weeks of differentiation in the presence or absence of xenogeneic substances. To quantify sialic acids (Neu5Gc and Neu5Ac) in hiPSC-CM, cell membranes were isolated and purified using PGC-SPE according to the method described in a previous report (21). The enriched sialic acids were analyzed using an Agilent 1290 Infinity LC system coupled with an Agilent 6495 triple quadrupole mass spectrometer. An Agilent AdvanceBio MS Spent Media column was used for LC separation with a binary gradient composed of solvent A (100% water and 10 mM ammonium formate (v/v) and solvent B (90% ACN with 10 mM ammonium formate (v/v) at a flow rate of 0.3 ml/min. N-linked glycans were released enzymatically from the prepared cell membranes by treating with PNGaseF and further enriched by solid-phase extraction (SPE), as previously described (22). Purified N-glycans were comprehensively analyzed using an Agilent 1260 Infinity LC and Agilent 6530 Q-TOF MS system. The abundance of each glycan was normalized to the total abundance of all observed glycans (23).

Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA). The standard unpaired Student’s t-test was used for comparisons between two groups. Analysis of variance followed by Tukey’s multiple comparisons test was used to compare more than two groups. RT-qPCR and cell biological assays were performed at least in triplicate with three to five independent experiments. Data are presented in terms of means±SEM. Statistical significance was set at p<0.05.

To compare the characteristics of hiPSC-CM depending on xenogeneic substances, we differentiated hiPSC into CM in the presence and absence of xenogeneic substances, such as Matrigel^TM, FBS, porcine trypsin, and bovine serum albumin. XC- and XF-hiPSC-CM were successfully generated using our modified differentiation method (Fig. 1). After the hiPSCs were expanded for 4 days on Matrigel^TM-coated plates (XC) or VTN-N-coated plates (XF), mesoderm induction occurred within 2 days after the media were changed under both XC and XF conditions. The addition of Matrigel^TM to the culture media enhanced cell attachment and mesoderm induction (XC conditions). However, differentiation into cardiac-progenitor cells occurred earlier upon VTN-N coating (XF conditions) (day 4) than under XC conditions (day 7). The expression of ISL1, a mesoderm marker, peaked at 2 weeks in both XC- and XF-hiPSC (Supplementary Fig. S1). The maturation of early-stage CM with contractile beating activity occurred more rapidly in the XF condition than in the XC condition. The contractile beating of hiPSC-CM with multiple contractile points was observed from 2 weeks after differentiation under both culture conditions, and synch-ronized beatings were observed at 4 weeks (Fig. 2A; Supplementary Video S1). After 4 weeks of differentiation, hiPSC-CM gathered and detached from the base of the plate, gradually forming a reticulated cardiac sheet. We continuously induced CM’s differentiation for further maturation into late-stage CM until 6 weeks. From these results, we demonstrated that the generation of XF-hiPSC-CMs with synergistic beating activities is robust and has even more rapid progress in differentiation from hiPSCs compared to XC-hiPSC-CM.

To evaluate the maturation status of XC- and XF-hiPSC-CM, we first measured cardiac-lineage mRNA expression using reverse transcription-quantitative PCR (RT-qPCR). The expression of myocardial sarcomeric protein-coding genes (MYH7, MYL2, ACTC1, and TNNT2) increased at 2 and 4 weeks after differentiation into cardiac-lineage cells under both conditions (Fig. 2B). At two weeks of differentiation, the fold changes in the expression of cardiac lineage-specific genes (MYH7, ACTC1, and TNNT2) increased in XF-hiPSC-CM compared to that in XC-hiPSC-CM (Fig. 2B, left panel).

The mRNA expression of cardiac ion channel-related genes, such as hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4) and ryanodine receptor 2 (RYR2), was significantly increased in both XC- and XF-hiPSC-CM compared with hiPSC. At two weeks of differentiation, RYR2 expression was significantly higher in XF-hiPSC-CM than in XC-hiPSC-CM (p<0.05, n=4) (Fig. 2B, right panel). In contrast, the expression of POU5F1, a marker of undifferentiated iPSC, was downregulated in both XC- and XF-hiPSC-CM after 2 to 4 weeks of differentiation. At 2 weeks, the expression of ISL1, a mesoderm marker, was higher in hiPSC-CM than in hiPSC; however, it was reduced by 4 weeks of differentiation under both XC and XF conditions (Supplementary Fig. S1). Next, we examined cardiac lineage-specific protein expression in hiPSC-CM. Immunocytochemistry confirmed the expression of cardiac sarcomere components and contractile cytoskeletal proteins, including ventricular myosin light chain (MLC-2v), alpha-smooth muscle actin (α-SMA), α-actinin, and cardiac troponin T (cTNT), in hiPSC-CM (Fig. 2C). These results demonstrated that the XF-hiPSC-CM shows characteristics of CMs with high expression of cardiac-specific genes and proteins, therefore suggesting differentiation into mature CMs at the molecular levels.

Prolonged culture after the induction of cardiac differentiation from stem cells can improve cardiac cell maturity with respect to morphology and electrophysiology (24-26). To compare the contractile functions of XF- and XC-hiPSC-CM, their electrophysiological properties were analyzed by using an in vitro multi-electrode array (MEA) system at 2 and 4 weeks of differentiation (Fig. 3). The representative field potential signals recorded from approximately 4 weeks of XC- and XF-hiPSC-CM were shown in Supplementary Fig. S2. In XC-hiPSC-CM, all analyzed field potential parameters increased significantly at 4 weeks from that at 2 weeks (Fig. 3A). The BPMs increased significantly from 33.3±2.38 bpm at 2 weeks to 52.77±4.08 bpm at 4 weeks (n=6, **p=0.0015). The FPA increased considerably, from 0.4±0.02 mV at 2 weeks to 1.56±0.15 mV at 4 weeks (n=6, ***p<0.0001). The FPDcF also increased from 319.8±11.27 ms at 2 weeks to 502.6±36.99 ms at 4 weeks (n=6, ***p=0.0008). In XF-hiPSC-CM, the beats per min (BPMs) increased significantly, from 43.2±1.52 bpm at 2 weeks to 52.4±2.6 bpm at 4 weeks (number of wells at each time point (n)=6, *p=0.0129). The field potential amplitude (FPA) also increased significantly, from 1.04±0.1 mV at 2 weeks to 2.01±0.17 mV at 4 weeks (n=6, ***p=0.0005). The field potential duration (FPDcF) did not change signifi-cantly, but showed an increasing trend, from 434.2±21.5 ms at 2 weeks to 466.6±18.07 ms at 4 weeks (Fig. 3B).

The electrophysiological parameters of hiPSC-CM were also compared to those of the commercial cardiomyocyte NexelTM Cardiosight^Ⓡ-S after 4 weeks of differentiation (Fig. 3C). Even though the FPA of Nexel^TM Cardiosight^Ⓡ-S was 4.28±0.4 mV, which was significantly higher than those of XF- or XC-hiPSC-CM (n=4, ***p<0.0001), the BPM and FPDcF of XF- or XC-hiPSC-CM did not differ significantly from those of Nexel^TM Cardiosight^Ⓡ-S. Con-versely, undifferentiated hiPSC cultured in XC or XF medium did not show electric excitability in the MEA system (data not shown). From the results of MEA analysis, we demonstrated that the XF-hiPSC-CMs also have mature electrophysiological characteristics of CMs, showing higher contractile activities compared to XC-hiPSC-CMs.

Based on the results of the characteristic analysis, both XC- and XF-hiPSC-CM exhibited characteristics of mature CM, with contractile function induced at 4 to 6 weeks of differentiation. Owing to safety concerns regarding the induction of a xenogeneic immune response, we investigated whether Neu5Gc, a xenogeneic sialic acid in humans, can be incorporated into terminal glycans in XC-hiPSC-CM. During the maintenance and cardiac-lineage differentiation of hiPSC into CM, we examined the Neu5Gc content in complete medium, conditioned medium, hiPSC lysates, and hiPSC-CM lysates using an indirect enzyme-linked immunosorbent assay (ELISA). For comparison of the XC/XF samples, FBS was used as the positive control and internal standard for Neu5Gc. As shown in Fig. 4A, Neu5Gc was detected in all XC media. The Neu5Gc content was significantly higher in the CM differentiation media (XCMdiff A and XCMdiff B) than in the hiPSC-culture media (mTeSR1) or cardiomyocyte maintenance media (XCM). In contrast, Neu5Gc was not detected in the XF media, such as the Essential 8 (E8) medium, CMdiff A, CMdiff B, and CM_main (Fig. 4A). Among the XC media obtained from the cell culture and differentiation experiments, the hiPSC-CM differentiation media showed a significantly higher Neu5Gc content (at 2 to 4 weeks vs. 0 weeks, n=3, *p<0.05) than the hiPSC culture media. However, Neu5Gc was not detected at any time point in the XF medium. Interestingly, we found that the incorporation of Neu5Gc into XC-hiPSC-CM was maintained during weeks 2 to 6 of differentiation (Fig. 4B). Using ELISA, we detected Neu5Gc, a non-human sialic acid form, in XC-hiPSC-CMs. As a safety concern, this important finding demonstrates that Neu5Gc can incorporate into hiPSC-CMs during the differentiation process from xenogeneic substances in the XC culture media.

To confirm the increase in the Neu5Gc content in XC-hiPSC-CM during differentiation, we further quantified Neu5Gc and Neu5Ac in hiPSC-CM during the time course of differentiation (0, 2, 4, and 6 weeks) using liquid chromatography-multiple reaction monitoring-mass spectrometry (LC/MRM MS). In both XC- and XF-hiPSC-CM, the levels of Neu5Ac, which is abundant in both humans and other mammals, were significantly higher at 2 to 6 weeks of differentiation than at 0 weeks (hiPSC) (Fig. 5). The total sialic acid content was calculated as the sum of the Neu5Ac and Neu5Gc contents. Neu5Gc was detected after 2 weeks of differentiation (>0.6 ng/μg protein) and maintained the amount during the differentiation of cells into XC-hiPSC-CM by 4 weeks (Fig. 5A, upper right panel). In contrast, Neu5Gc was not detected in XF-hiPSC-CM at any time point (Fig. 5B, lower right panel). To confirm whether Neu5Gc present in the XC differentiation media was incorporated at the terminal position of N-linked glycans in hiPSC-CM, we further analyzed the N-linked glycan profiles of hiPSC-CM. Terminal Neu5Gc was detected on the glycan residues, which were separated from XC-hiPSC-CM during 2 to 6 weeks of differentiation. Neu5Gc was not detected in the glycan residues of XF-hiPSC-CM (Fig. 6). In this experiment using LC/MRM MS, the results of specific quantification of Neu5Ac/Neu5Gc confirmed the results of Neu5Gc detection by ELISA analysis. Moreover, from the results of glycan profiling analysis, the quantified amounts of Neu5Gc are derived from the XC-hiPSC-CM during the differentiation process, but not from the media itself.