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N-glycans: phenotypic homology and structural differences between myocardial cells and induced pluripotent stem cell-derived cardiomyocytes.

Kawamura T, Miyagawa S, Fukushima S, Yoshida A, Kashiyama N, Kawamura A, Ito E, Saito A, Maeda A, Eguchi H, Toda K, Lee JK, Miyagawa S, Sawa Y - PLoS ONE (2014)

Bottom Line: The quantity of high-mannose type (immature) N-glycans on the iPSCs decreased with cardiomyogenic differentiation, but did not reach the low levels observed in the heart.We observed a similar reduction in neutral N-glycans and an increase in fucosylated or sialyl N-glycans.The expression of N-glycan on murine iPSCs changed toward the myocardial phenotype during cardiomyogenic differentiation, leaving the structural differences of NeuGc content or Galα1-6 Gal structures.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.

ABSTRACT
Cell surface glycans vary widely, depending on cell properties. We hypothesized that glycan expression on induced pluripotent stem cells (iPSCs) might change during cardiomyogenic differentiation toward the myocardial phenotype. N-glycans were isolated from iPSCs, iPSC-derived cardiomyocytes (iPSC-CM), and original C57BL/6 mouse myocardium (Heart). Their structures were analyzed by a mapping technique based on HPLC elution times and MALDI-TOF/MS spectra. Sixty-eight different N-glycans were isolated; the structures of 60 of these N-glycans were identified. The quantity of high-mannose type (immature) N-glycans on the iPSCs decreased with cardiomyogenic differentiation, but did not reach the low levels observed in the heart. We observed a similar reduction in neutral N-glycans and an increase in fucosylated or sialyl N-glycans. Some structural differences were detected between iPSC-CM and Heart. No N-glycolyl neuraminic acid (NeuGc) structures were detected in iPSC-CM, whereas the heart contained numerous NeuGc structures, corresponding to the expression of cytidine monophosphate-N-acetylneuraminic acid hydroxylase. Furthermore, several glycans containing Galα1-6 Gal, rarely identified in the other cells, were detected in the iPSC-CM. The expression of N-glycan on murine iPSCs changed toward the myocardial phenotype during cardiomyogenic differentiation, leaving the structural differences of NeuGc content or Galα1-6 Gal structures. Further studies will be warranted to reveal the meaning of the difference of N-glycans between the iPSC-CM and the myocardium.

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Cardiomyogenic differentiation of iPSCs and cardiomyocyte purification.(a) The cardiomyogenic differentiation protocol and cardiomyocyte purification process are illustrated. (b) IPSC-CMs stained with anti-α-actinin antibody (Alexa Fluor 488), anti-troponin I (Alexa Fluor 594) and DAPI, were analyzed with a confocal laser scanning microscopy. Abbreviations: EB, embryonic body; MEM, Modified Eagle's Medium; DMEM, Dulbecco's Modified Eagle's Medium; BIO, 6-bromoindirubin-3′-oxime.
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pone-0111064-g001: Cardiomyogenic differentiation of iPSCs and cardiomyocyte purification.(a) The cardiomyogenic differentiation protocol and cardiomyocyte purification process are illustrated. (b) IPSC-CMs stained with anti-α-actinin antibody (Alexa Fluor 488), anti-troponin I (Alexa Fluor 594) and DAPI, were analyzed with a confocal laser scanning microscopy. Abbreviations: EB, embryonic body; MEM, Modified Eagle's Medium; DMEM, Dulbecco's Modified Eagle's Medium; BIO, 6-bromoindirubin-3′-oxime.

Mentions: Cardiomyogenic differentiation of the iPSCs was performed as described [20], [21], with modifications, followed by purification with glucose-free medium supplemented with lactic acid [22]; iPSCs (3 × 103) were resuspended in 100-µL aliquots of differentiation medium [DM; Dulbecco's Modified Eagle's Medium (DMEM; Nacalai Tesque) containing 15% fetal bovine serum (FBS; Biofill), 100 µmol/L non-essential amino acids (NEAA; Invitrogen), 2 mmol/L L-glutamine (Invitrogen), and 0.1 mmol/L 2-mercaptoethanol (Invitrogen)] containing 0.2 µmol/L 6-bromoindirubin-3′-oxime (BIO; a glycogen synthase kinase-3β inhibitor, to activate the Wnt-signaling pathway) (Calbiochem), and cultured in 96-well Corning Costar Ultra-Low attachment multiwell plates (Sigma-Aldrich) for 3 days. On day 3, an additional 100 µL DM without BIO was added to each well. On day 5, individual embryoid bodies (EBs) were transferred to 100-mm gelatin-coated dishes (250 EBs per dish). On days 6, 7, 10, 11, 14, and 15 the medium was exchanged for serum-free Modified Eagle's Medium (MEM; Invitrogen) with insulin transferrin-selenium-X (Invitrogen). On days 8, 9, 12, and 13, the medium was exchanged for Glucose-free DMEM (no glucose, no pyruvate, Invitrogen) supplemented with 4 mmol/L lactic acid (Wako Pure Chemical) for purification of cardiomyocytes. On day 16, the contracting cell clusters were used as cardiomyogenically differentiated iPSCs (959A2-1 CMs, 959C1-1 CMs, 956F-1 CMs: iPSC-CMs). The protocol and purification process are illustrated in Figure 1a.


N-glycans: phenotypic homology and structural differences between myocardial cells and induced pluripotent stem cell-derived cardiomyocytes.

Kawamura T, Miyagawa S, Fukushima S, Yoshida A, Kashiyama N, Kawamura A, Ito E, Saito A, Maeda A, Eguchi H, Toda K, Lee JK, Miyagawa S, Sawa Y - PLoS ONE (2014)

Cardiomyogenic differentiation of iPSCs and cardiomyocyte purification.(a) The cardiomyogenic differentiation protocol and cardiomyocyte purification process are illustrated. (b) IPSC-CMs stained with anti-α-actinin antibody (Alexa Fluor 488), anti-troponin I (Alexa Fluor 594) and DAPI, were analyzed with a confocal laser scanning microscopy. Abbreviations: EB, embryonic body; MEM, Modified Eagle's Medium; DMEM, Dulbecco's Modified Eagle's Medium; BIO, 6-bromoindirubin-3′-oxime.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214687&req=5

pone-0111064-g001: Cardiomyogenic differentiation of iPSCs and cardiomyocyte purification.(a) The cardiomyogenic differentiation protocol and cardiomyocyte purification process are illustrated. (b) IPSC-CMs stained with anti-α-actinin antibody (Alexa Fluor 488), anti-troponin I (Alexa Fluor 594) and DAPI, were analyzed with a confocal laser scanning microscopy. Abbreviations: EB, embryonic body; MEM, Modified Eagle's Medium; DMEM, Dulbecco's Modified Eagle's Medium; BIO, 6-bromoindirubin-3′-oxime.
Mentions: Cardiomyogenic differentiation of the iPSCs was performed as described [20], [21], with modifications, followed by purification with glucose-free medium supplemented with lactic acid [22]; iPSCs (3 × 103) were resuspended in 100-µL aliquots of differentiation medium [DM; Dulbecco's Modified Eagle's Medium (DMEM; Nacalai Tesque) containing 15% fetal bovine serum (FBS; Biofill), 100 µmol/L non-essential amino acids (NEAA; Invitrogen), 2 mmol/L L-glutamine (Invitrogen), and 0.1 mmol/L 2-mercaptoethanol (Invitrogen)] containing 0.2 µmol/L 6-bromoindirubin-3′-oxime (BIO; a glycogen synthase kinase-3β inhibitor, to activate the Wnt-signaling pathway) (Calbiochem), and cultured in 96-well Corning Costar Ultra-Low attachment multiwell plates (Sigma-Aldrich) for 3 days. On day 3, an additional 100 µL DM without BIO was added to each well. On day 5, individual embryoid bodies (EBs) were transferred to 100-mm gelatin-coated dishes (250 EBs per dish). On days 6, 7, 10, 11, 14, and 15 the medium was exchanged for serum-free Modified Eagle's Medium (MEM; Invitrogen) with insulin transferrin-selenium-X (Invitrogen). On days 8, 9, 12, and 13, the medium was exchanged for Glucose-free DMEM (no glucose, no pyruvate, Invitrogen) supplemented with 4 mmol/L lactic acid (Wako Pure Chemical) for purification of cardiomyocytes. On day 16, the contracting cell clusters were used as cardiomyogenically differentiated iPSCs (959A2-1 CMs, 959C1-1 CMs, 956F-1 CMs: iPSC-CMs). The protocol and purification process are illustrated in Figure 1a.

Bottom Line: The quantity of high-mannose type (immature) N-glycans on the iPSCs decreased with cardiomyogenic differentiation, but did not reach the low levels observed in the heart.We observed a similar reduction in neutral N-glycans and an increase in fucosylated or sialyl N-glycans.The expression of N-glycan on murine iPSCs changed toward the myocardial phenotype during cardiomyogenic differentiation, leaving the structural differences of NeuGc content or Galα1-6 Gal structures.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.

ABSTRACT
Cell surface glycans vary widely, depending on cell properties. We hypothesized that glycan expression on induced pluripotent stem cells (iPSCs) might change during cardiomyogenic differentiation toward the myocardial phenotype. N-glycans were isolated from iPSCs, iPSC-derived cardiomyocytes (iPSC-CM), and original C57BL/6 mouse myocardium (Heart). Their structures were analyzed by a mapping technique based on HPLC elution times and MALDI-TOF/MS spectra. Sixty-eight different N-glycans were isolated; the structures of 60 of these N-glycans were identified. The quantity of high-mannose type (immature) N-glycans on the iPSCs decreased with cardiomyogenic differentiation, but did not reach the low levels observed in the heart. We observed a similar reduction in neutral N-glycans and an increase in fucosylated or sialyl N-glycans. Some structural differences were detected between iPSC-CM and Heart. No N-glycolyl neuraminic acid (NeuGc) structures were detected in iPSC-CM, whereas the heart contained numerous NeuGc structures, corresponding to the expression of cytidine monophosphate-N-acetylneuraminic acid hydroxylase. Furthermore, several glycans containing Galα1-6 Gal, rarely identified in the other cells, were detected in the iPSC-CM. The expression of N-glycan on murine iPSCs changed toward the myocardial phenotype during cardiomyogenic differentiation, leaving the structural differences of NeuGc content or Galα1-6 Gal structures. Further studies will be warranted to reveal the meaning of the difference of N-glycans between the iPSC-CM and the myocardium.

Show MeSH
Related in: MedlinePlus