Figure 1.
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.
Figure 2.
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.
Figure 3.
Ca2+ transient measurement of iPSC-CMs pre and post the administration of isoproterenol.
(a) Fluo-8 loaded iPSC-CMs at the time of low (left) and high (right) fluorescence. (b), (c) Sequentially measured fluorescence intensity of Fluo-8 loaded iPSC-CMs pre (b) and post (c) the administration of 1 µM isoproterenol.
Figure 4.
Anion-exchange DEAE elution profiles of PA-glycans.
PA-glycans were fractionated according to their sialic acid content as neutral (peak 1), monosialyl (peak 3), and disialyl (peak 4) oligosaccharide fractions. Peaks 2 and 5 represent fractions containing no detectable PA-oligosaccharides.
Figure 5.
Reverse-phase ODS elution profiles of the neutral PA-glycans.
The neutral fractions were individually applied to the ODS column and eluted according to their hydrophobicity. N1′, N2′, N4′, N5′ and N11′: epimerization of N1, N2, N4, N5 and N11. *Fractions containing no detectable PA-oligosaccharides.
Figure 6.
Reverse-phase ODS elution profiles of monosialyl PA-glycans.
The monosialyl fractions were individually applied to the ODS column and eluted according to their hydrophobicity. M2′, M3′, M4′, M5′, M7′, M8′, M18′, M19′, M22′, M24′ and M25′: epimerization of M2, M3, M4, M5, M7, M8, M18, M19, M22, M24 and M25. *Fractions containing no detectable PA-oligosaccharides. ‡Magnification ratio to the fluorescence intensity of asialoglycan of each sample.
Figure 7.
Reverse-phase ODS elution profiles of disialyl PA-glycans.
The disialyl fractions were individually applied to the ODS column and eluted according to their hydrophobicity. D1′, D4′, D10′ and D12′: epimerization of D1, D4, D10 and D12; *Fractions containing no detectable PA-oligosaccharides. ‡Magnification ratio to the fluorescence intensity of asialoglycan of each sample.
Figure 8.
Structures of neutral, monosialyl, and disialyl PA-oligosaccharides in iPSCs, iPSC-CM, and heart cells.
Glucose units (GU) were calculated from the peak elution times for the ODS column in Figure 5, 6 and 7, and the amide column (data not shown). Average mass (Mass) calculated from the mlz values of [M+Na]+ or [M+H]+ ion for neutral, [M-H]− ion for monosialyl, and [M-H]− & [M+Na-2H]− ions for disialyl PA-oligosaccharides.
Figure 9.
Relative quantities of neutral, monosialyl, and disialyl PA-oligosaccharides in iPSCs, iPSC-CM, and heart cells.
Relative quantities of each glycan, calculated from the peak area in Figure 5, 6 and 7 vs. total N-glycan content in each cell, were expressed in the doughnut charts. Relative quantities of the asialoglycans, the monosialoglycans and the disialoglycans were showed outside of the charts, and relative quantities of the high mannose type glycans were showed inside of the charts. Asialoglycan (AS): the total volume of N1-N17; Monosialoglycan (MS): the total volume of M1-M23; Disialoglycan (DS): the total volume of D1-D12, High mannose-type glycan (HM): the total volume of N1–N6-1, N6-2, N7.
Figure 10.
Rarely expressed NeuGc-containing glycans in iPSCs and iPSC-CMs.
(a) Relative quantities of NeuAc- and NeuGc-containing glycans; Monosialoglycans containing NeuAc and Disialoglycans containing two NeuAc (A, A/A): the total volume of M1, M2-1, M2-2, M5-M8, M10-M14, M16-M19, M20-2, M21-M23, D4-1, D4-2, D6, D7, D9, D10-1, D12, Disialoglycan containing NeuAc and NeuGc (A/G): D11, Monosialoglycan containing NeuGc and Disialoglycan containing two NeuGc (G, G/G): the total volume of M2-3, M3, M4, M9, M15, M20-1, D1-D3, D5-1, D5-2, D8, D10-2. (b) Transcript expression of ST3Gal-III, ST3Gal-IV, ST6Gal-I, and CMAH; Transcript expression of glycosyltransferases in iPSCs, iPSC-CM, and heart cells was analyzed by real-time PCR. Results are expressed as the mean ± standard deviation. *P<0.05 vs. Heart, †P<0.05 vs. iPSC-CM (all of the 959A2-1 CM, 959C1-1 CM and 956F-1 CM).