The Nglycans of N9-3, M8, M12, M17, and M23, which were identified after a-galactosidase digestion, contained Gala1-6Gal, not only in the neutral glycans but also in the monosialyl N-glycans of the iPSC-CM preparation. The same structure was not found in iPSCs, but only one structure, M23, was present in Heart cells. Therefore, in iPSC-CMs, Gala1-6Gal enzyme activity appears to be up-regulated in comparison to wild-type myocardium, although enzyme activity was not assessed by RT-PCR because of the limited availability of genetic sequence data. The D8 was identified in all of three iPSC lines and not in the iPSC-CMs and Heart. This structure, unfortunately not identified in this study, may be R428 useful as markers of undifferentiated iPSCs in the same way as well-known pluripotency biomarkers such as stage-specific embryonic antigens -3, SSEA-4. Previous MALDI-TOF/MS and MS/MS studies concluded that many kinds of N-glycans are found in organs and cells. The number of detected N-glycans is attributed to the sensitivity of the MS and HPLC methods employed. That is, MS data are sensitive and can be rapidly obtained, but a glycan structure is identified based only on the calculated molecular weight. Therefore, discriminating between isomeric structures is difficult. On the other hand, it thus appears that the accuracy of the data presented here using HPLC mapping in conjunction with a MALDI-TOF technique provides much more detailed information. Our data were used to identify the representative features of each N-glycan in these three cell types. There may be a concern that the heart tissue used in this study contains connective tissues, vessels or nerves other than cardiomyocytes. Therefore, some of the N-glycans detected from the Heart sample might be derived from the tissues other than cardiomyocytes. However, heart is majority composed by cardiomyocytes, and furthermore, even if a small amount of N-glycans derived from connective tissues were contaminated in the Heart sample, the main evidences in this study, such as the proportion of the high-mannose type N-glycans, the ratio of the active sialyltransferase genes, the existence of NeuGc, and the uncommonness of Gala1-6 Gal, are essentially not affected. In summary, murine iPSCs were rich in high-mannose type Nglycans but very poor in sialyl type N-glycans. Murine heart tissue contained a relatively low volume of high-mannose glycans, but was very rich in neuraminic acid, especially NeuGc type sialyl structures. Under these conditions, the volume of each type of glycan was similar for iPSC-CMs and iPSCs. That is, they were rich in high-mannose and relatively poor in sialyl type N-glycans by volume. In addition, most of the sialyl structures of the iPSCCMs were different from those of the Heart, and the iPSC-CMs expressed no NeuGc. Moreover, the iPSC-CMs produced several unique glycans with the Gala1-6Gal structure. These results provide important data that can be useful in future clinical iPSC studies. In this study, for deeply understanding the relationship between the N-glycan expression and cardiomyogenic differentiation. Knockout or knock-down of the genes related to cardiomyogenic differentiation or glycosylation may be useful for such purpose. However, the N-glycan signature in the cell surface is determined by a variety of the genes. Knock-out or knock-down of a single gene related to cardiomyogenic differentiation would alter an array of gene expressions, such as sarcomere proteins, transcriptional factors, or cell surface proteins, all of which would affect the signature of N-glycans in the cell surface.