• International Journal of Stem Cells
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• v.16 no.3
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• pp.281-292
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• 2023

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.

• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.219-219
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• 2003

• Bulletin of the Korean Chemical Society
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• v.35 no.3
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• pp.955-957
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• 2014

• 한국생물공학회:학술대회논문집
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• 2005.10a
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• pp.424-425
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• 2005

• Proceedings of the Korean Society of Sericultural Science Conference
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• 2004.10a
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• pp.78-78
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• 2004

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2004.06a
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• pp.278-281
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• 2004

The $\alpha$1,6-mannosyltransferase encoded by Saccharomyces cerevisiae OCH1 plays a key role for the outer chain initiation of the N-linked oligosaccharides. A search for Hansenula polymorpha genes homologous to S. cerevisiae OCHI (ScOCH1) has revealed seven open reading frames (ORF100, ORF142, ORF168, ORF288, ORF379, ORF576, ORF580). All of the seven ORFs are predicted to be a type II integral membrane protein containing a transmembrane domain near the amino-terminal region and has a DXD motif, which has been found in the active site of many glycosyltransferases. Among this seven-membered OCH1 gene family of H. polymorpha, we have carried out a functional analysis of H. polymorpha ORF168 (HpOCH2) showing the highest identity to ScOCH1. Inactivation of this protein by disruption of corresponding gene resulted in several phenotypes suggestive of cell wall defects, including hypersensitivity to hygromycin B and sodium deoxycholate. The structural analysis of N-glycans synthesized in HpOCH2-disrupted strain (Hpoch2Δ) and the in vitro $\alpha$1,6-mannosyltransferase activity assay strongly indicate that HpOch2p is a key enzyme adding the first $\alpha$1,6-mannose residue on the core glycan Man$_{8}$GlcNAc$_2$. The Hpoch2Δ was further genetically engineered to synthesize a recombinant glycoprotein with the human compatible N-linked oligosaccharide, Man$_{5}$GlcNAc$_2$, by overexpression of the Aspergillus saitoi $\alpha$1,2-mannosidase with the 'HDEL” ER retention signal.gnal.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.2
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• pp.691-695
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• 2016

Protein glycosylation is the most common posttranslational modification in mammalian cells. Aberrant protein glycosylation has been reported in various diseases, including cancer. We identified and quantified the glycan structures of O-linked glycoprotein from cholangiocarcinoma (CCA) cell lines from different histological types and compared their profiles by nanospray ionization-linear ion trap mass spectrometry (NSI-$MS^n$). Five human CCA cell lines, K100, M055, M139, M213 and M214 were characterized. The results showed that the O-linked glycans of the CCA cell lines comprised tri- to hexa-saccharides with terminal galactose and sialic acids: NeuAc1Gal1GalNAc1, Gal2GlcNAc1GalNAc1, NeuAc2Gal1GalNAc1 NeuAc1Gal2GlcNAc1GalNAc1 and NeuAc2Gal2GlcNAc1GalNAc1 All five CCA cell lines showed a similar glycan pattern, but with differences in their quantities. NeuAc1Gal1GalNAc1 proved to be the most abundant structure in poorly differentiated adenocarcinoma (K100; 57.1%), moderately differentiated adenocarcinoma (M055; 42.6%) and squamous cell carcinoma (M139; 43.0%), while moderately to poorly differentiated adenocarcinoma (M214; 40.1%) and adenosquamous cell carcinoma (M213; 34.7%) appeared dominated by $NeuA_{c2}Gal_1GalNA_{c1}$. These results demonstrate differential expression of the O-linked glycans in the different histological types of CCA. All five CCA cell lines have abundant terminal sialic acid (NeuAc) O-linked glycans, suggesting an important role for sialic acid in cancer cells. Our structural analyses of glycans may provide important information regarding physiology of disease-related glycoproteins in CCA.

• Journal of Microbiology and Biotechnology
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• v.18 no.2
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• pp.383-391
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• 2008

The glycans linked to the insect cell-derived glycoproteins are known to differ from those expressed in mammalian cells, partly because of the low level or lack of glycosyltransferase activities. GnT II, GnT IV, GnT V, and ST3Gal IV, which play important roles in the synthesis of tetraantennarytype complex glycan structures in mammalian cells, were overexpressed in Trichoplusia ni cells by using a baculovirus expression vector. The glycosyltransferases, expressed as a fusion form with the IgG-binding domain, were secreted into the culture media and purified using IgG sepharose resin. The enzyme assay, performed using a pyridylaminated-sugar chain as an acceptor, indicated that the purified glycosyltransferases retained their enzyme activities. Human erythropoietin expressed in T. ni cells (rhEPO) was subjected to in vitro glycosylation by using recombinant glycosyltransferases and was converted into complex-type glycan with terminal sialic acid. The presence of Nacetylglucosamine, galactose, and sialic acid on the rhEPO moiety was detected by a lectin blot analysis, and the addition of galactose and sialic acid to rhEPO was confirmed by autoradiography using $UDP-^{14}C-Gal\;and\;CMP-^{14}C-Sia$ as donors. The in vitro glycosylated rhEPO was injected into mice, and the number of reticulocytes among the ed blood cells was counted using FACS. A significant increase in the number of reticulocytes was not observed in the mice injected with in vitro glycosylated rhEPO as compared with those injected with rhEPO.

• KSBB Journal
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• v.31 no.4
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• pp.270-276
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• 2016

In glycoprotein, Terminal sialic acid residues of N-linked glycan are imperative things because they prevent the recognition from asialoglycoprotein-receptor that affect the half-life of glycoproteins. So establishment of culture process for enhancing sialic acid is important to maximize sialic acid contents of glycoprotein. In this study, we investigated effects of biphasic culture of Chinese hamster ovary (CHO) cells producing albumin-erythropoietin to increase sialylation. Biphasic cultures were performed with shift of $CO_2$ concentrations and temperatures at day 5 when viable cell density was decreased and sialidase was started to be released by cell lysis. The examined temperature set points were 33, 35 and $37^{\circ}C$ respectively and the $CO_2$ concentration was 1, 5, 10 and 15%. We confirmed that sialidase activity was the lowest in biphasic culture that was shifted from normal culture condition to 1% of $CO_2$ and $33^{\circ}C$ on day 5. However, the temperature and concentration of $CO_2$ have little effect on activity of ${\alpha}2,3$-sialyltransferase. Also, sialic acid contents were enhanced 1.13-fold higher than that in control culture. In conclusion, Biphasic cultivation in CHO cells led to inhibition of sialidase activity and increases of sialylated glycan.

• KSBB Journal
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• v.32 no.3
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• pp.193-198
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• 2017

Sialylation is important in producing therapeutic proteins such as antibody, cytokine and fusion protein. Thus, enhancement of sialylation is usually performed in CHO cell cultures. ${\alpha}2,6$-Sialyltransferase (ST), which plays a key role in the attachment of ${\alpha}2,6-sialic$ acid, is present in human cells but not in Chinese hamster ovary (CHO) cells. Overexpression of ${\alpha}2,6-ST$ can be used for enhancing the degree of sialylation and achieving human-like glycosylation. In this study, we constructed CHO cells producing human cytotoxic T-lymphocyte antigen4-immunoglobulin (hCTLA4-Ig) as well as ${\alpha}2,6-ST$. Transfected CHO cells were selected using G418 and stable cell line was established. Profiles of viable cell density and hCTLA4-Ig titer in an overexpressed cell line were similar to those of a wild-type cell line. It was confirmed that the total amount of sialic acid was increased and ${\alpha}2,6-sialic$ acid was attached to the terminal residues of N-glycan of hCTLA4-Ig by ESI-LC-MS. Compared to 100% of ${\alpha}2,3-sialic$ acid in wild type cells, 70.9% of total sialylated N-glycans were composed of ${\alpha}2,6-sialic$ acid in transfected cells. In conclusion, overexpression of ${\alpha}2,6-ST$ in CHO cells led to the increase of both the amount of total sialylated N-glycan and the content of ${\alpha}2,6-sialic$ acid, which is more resemble to human-like structure of glycosylation.