Int J Stem Cells.  2023 Aug;16(3):281-292. 10.15283/ijsc22158.

Effect of Xenogeneic Substances on the Glycan Profiles and Electrophysiological Properties of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

• Kim YG
• Yun JH
• Park JW
• Seong D
• Lee Sh
• Park KD
• Lee HA
• Park M

• KMID: 2545228
• DOI: http://doi.org/10.15283/ijsc22158

Abstract

Background and Objectives
Human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte (CM) hold great promise as a cellular source of CM for cardiac function restoration in ischemic heart disease. However, the use of animal-derived xenogeneic substances during the biomanufacturing of hiPSC-CM can induce inadvertent immune responses or chronic inflammation, followed by tumorigenicity. In this study, we aimed to reveal the effects of xenogeneic substances on the functional properties and potential immunogenicity of hiPSC-CM during differentiation, demonstrating the quality and safety of hiPSC-based cell therapy.
Methods and Results
We successfully generated hiPSC-CM in the presence and absence of xenogeneic substances (xeno-containing (XC) and xeno-free (XF) conditions, respectively), and compared their characteristics, including the contractile functions and glycan profiles. Compared to XC-hiPSC-CM, XF-hiPSC-CM showed early onset of myocyte contractile beating and maturation, with a high expression of cardiac lineage-specific genes (ACTC1, TNNT2, and RYR2) by using MEA and RT-qPCR. We quantified N-glycolylneuraminic acid (Neu5Gc), a xenogeneic sialic acid, in hiPSC-CM using an indirect enzyme-linked immunosorbent assay and liquid chromatography-multiple reaction monitoring-mass spectrometry. Neu5Gc was incorporated into the glycans of hiPSC-CM during xeno-containing differentiation, whereas it was barely detected in XF-hiPSC-CM.
Conclusions
To the best of our knowledge, this is the first study to show that the electrophysiological function and glycan profiles of hiPSC-CM can be affected by the presence of xenogeneic substances during their differentiation and maturation. To ensure quality control and safety in hiPSC-based cell therapy, xenogeneic substances should be excluded from the biomanufacturing process.

Keyword

hiPSC; Cardiomyocytes; Xenogeneic substances; N-glycolylneuraminic acid; Quality control; Cell therapy

Figure

• 

  Fig. 1 Schematic representation of the protocol for the differentiation of hiPSC into CM. (A) In the XC culture condition, hiPSC-CM were generated on MatrigelTM-coated plates according to the modified GiAB (MM.1) protocol. Up to 5 days after the differentiation into CM commenced, 1~1.5 vol% MatrigelTM was added to the medium. (B) In the XF culture condition, hiPSC were differentiated into cardiac-lineage cells on VTN-N-coated 12-well culture plates using the PSC Cardiomyocyte Differentiation Kit.

• 

  Fig. 2 Analysis of the characteristics of hiPSC-CM. (A) Microscopic image of cells of cardiac lineage at 1, 2, and 4 weeks after the differentiation of hiPSC into CM under XC and XF conditions. (B) mRNA expressions of cardiac lineage-specific marker genes in XC- and XF-hiPSC-CM were compared at 0, 2, and 4 weeks post-differentiation using reverse transcription-quantitative PCR. The expression values were normalized against that of 18S rRNA as a housekeeping gene. n=3 per group, *p<0.05, XC-hiPSC-CM vs. XF-hiPSC-CM using unpaired Student’s t-test. (C) Protein expression of cardiac lineage-specific markers was determined by immunostaining at 4 weeks after differentiation under the XF condition. MLC-2v, myosin light chain-2v (ventricular/cardiac muscle isoform) (red); cTnT, cardiac troponin T (green); α-SMA, α-smooth muscle actin (red); α-actinin, a cytoskeletal actin-binding protein (green). Cell nuclei were counterstained using DAPI (blue).

• 

  Fig. 3 Contractile properties of CM that differentiated from hiPSC. The electrophysiological properties of hiPSC-CM were assessed using a multi-electrode array (MEA) assay. Differences in the field potential parameters of (A) XC-hiPSC-CM and (B) XF-hiPSC-CM at 2 and 4 weeks after differentiation. *p<0.05, **p<0.01, ***p<0.001. (C) Comparison of the field potential parameters of XC-hiPSC-CM and XF-hiPSC-CM at 4 weeks. The commercially available CM NexelTM Cardiosight®-S was used as a positive control. n=6 per group of XC/XF-CM, n=4 for NexelTM Cardiosight®-S. ***p<0.001 by one-way analysis of variance with Tukey’s multiple comparisons test. Val-ues are presented in terms of means± SEM. BPM, beats per minute; FPA, field potential amplitude; FPDcF, field potential duration corrected with Fride-ricia's formula.

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  Fig. 4 Evaluation of immunogenic Neu5Gc in hiPSC-CM using an indirect ELISA. (A) Neu5Gc was only detected in XC complete media. (B) Neu5Gc expression in the XC-conditioned media and XC-hiPSC-CM lysates was significantly higher at 2 to 4 weeks of differentiation than at 0 weeks (hiPSC). Neu5Gc was not detected in XF media and XF-hiPSC-CM lysates. Fetal bovine serum was used as a positive control and experimental internal control. Statistical analysis was performed using analysis of variance, followed by Tukey’s multiple comparisons test. Values are presented in terms of means±SEM. **p<0.01, ***p<0.001. n=3 per group.

• 

  Fig. 5 Quantification of Neu5Gc in the hiPSC-CM lysate using LC/MRM MS. The total sialic acid content (Ne-u5Ac and Neu5Gc) was significantly higher at 2 to 6 weeks (W) after differentiation into hiPSC-CM than at 0 weeks (hiPSCs) under both (A) XC condition and (B) XF conditions. Neu5Gc was only detected in XC-hiPSC-CM, even though the most prominent increase in the level of sialic acid was attributed to Neu5Ac. Statistical analysis was performed using the Student’s t-test. *p<0.05, ***p<0.001. Values are presented in terms of means±SEM. n=3 per group. N.D., not detected.

• 

  Fig. 6 Sialic acid residues on the N-linked glycans of hiPSC-CM. The N-linked glycan structures are indicated by each major peak with the acquisition time. (A) Terminal Neu5Gc was detected on the glycans that were separated from XC-hiPSC-CM during 2 to 6 weeks of differentiation (indicated in the blue box). (B) Neu5Gc was not detected in the glycan residues of XF-hiPSC-CM. 0W, hiPSC; 2-6W, hiPSC-CM.

Reference

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