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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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Highly purified iPSC-CMs expressing cardiomyocyte marker genes.(a) Transcript expression of Nkx2.5, αMHC, ANP and Isl1 in the iPSCs and the iPSC-CMs were analyzed by real-time PCR. Results are expressed as the mean ± standard deviation. *P<0.05. (b) IPSC-CMs and iPSCs stained with anti-troponin T antibody or the isotype control, followed by Alexa Fluor 488-conjugated anti-mouse IgG antibody, were analyzed by flow cytometry.
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pone-0111064-g002: Highly purified iPSC-CMs expressing cardiomyocyte marker genes.(a) Transcript expression of Nkx2.5, αMHC, ANP and Isl1 in the iPSCs and the iPSC-CMs were analyzed by real-time PCR. Results are expressed as the mean ± standard deviation. *P<0.05. (b) IPSC-CMs and iPSCs stained with anti-troponin T antibody or the isotype control, followed by Alexa Fluor 488-conjugated anti-mouse IgG antibody, were analyzed by flow cytometry.

Mentions: Cardiomyogenic differentiation was induced in murine iPSCs by using a slightly modified culture protocol (Figure 1a). The iPSC-CMs showed significantly higher expressions of Nkx2.5, αMHC, ANP and Isl1 than undifferentiated iPSCs by semi-quantitative real-time PCR (Figure 2a), and showed sarcomere structures observed by immunohistological staining of α-actinin and troponin I (Figure 1b). The iPSC-CMs were functional with Ca2+ transient measurement (Figure 3a, b) and their beating rates were increased by the administration of isoproterenol (Figure 3c), meaning they had β-adrenergic receptors. Nearly all of the iPSC-CMs exhibited spontaneous and regular beating at room temperature (Video S1). The differentiation efficiency of murine iPSC was evaluated by flow cytometry analysis. More than 95% of the 959A2-1 CMs, 92% of the 959C1-1 CMs and 90% of the 956F-1 CMs were positive for troponin T (Figure 2b), while the undifferentiated iPSCs rarely expressed troponin T (Figure 2b).


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)

Highly purified iPSC-CMs expressing cardiomyocyte marker genes.(a) Transcript expression of Nkx2.5, αMHC, ANP and Isl1 in the iPSCs and the iPSC-CMs were analyzed by real-time PCR. Results are expressed as the mean ± standard deviation. *P<0.05. (b) IPSC-CMs and iPSCs stained with anti-troponin T antibody or the isotype control, followed by Alexa Fluor 488-conjugated anti-mouse IgG antibody, were analyzed by flow cytometry.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111064-g002: Highly purified iPSC-CMs expressing cardiomyocyte marker genes.(a) Transcript expression of Nkx2.5, αMHC, ANP and Isl1 in the iPSCs and the iPSC-CMs were analyzed by real-time PCR. Results are expressed as the mean ± standard deviation. *P<0.05. (b) IPSC-CMs and iPSCs stained with anti-troponin T antibody or the isotype control, followed by Alexa Fluor 488-conjugated anti-mouse IgG antibody, were analyzed by flow cytometry.
Mentions: Cardiomyogenic differentiation was induced in murine iPSCs by using a slightly modified culture protocol (Figure 1a). The iPSC-CMs showed significantly higher expressions of Nkx2.5, αMHC, ANP and Isl1 than undifferentiated iPSCs by semi-quantitative real-time PCR (Figure 2a), and showed sarcomere structures observed by immunohistological staining of α-actinin and troponin I (Figure 1b). The iPSC-CMs were functional with Ca2+ transient measurement (Figure 3a, b) and their beating rates were increased by the administration of isoproterenol (Figure 3c), meaning they had β-adrenergic receptors. Nearly all of the iPSC-CMs exhibited spontaneous and regular beating at room temperature (Video S1). The differentiation efficiency of murine iPSC was evaluated by flow cytometry analysis. More than 95% of the 959A2-1 CMs, 92% of the 959C1-1 CMs and 90% of the 956F-1 CMs were positive for troponin T (Figure 2b), while the undifferentiated iPSCs rarely expressed troponin T (Figure 2b).

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