br Materials and methods br Results br Discussion Recent neu

Materials and methods
Results
Discussion Recent neurogenesis studies and the discovery of multipotent neural stem Fostriecin sodium salt cost derived from the adult mammalian CNS, including that of humans, have had a considerable influence on potential treatments for neural injury (Gross, 2000; Ming and Song, 2005). Although iPSCs are promising candidates as a cell source for applications in neural regeneration (Takahashi et al., 2007; Takahashi and Yamanaka, 2006), little is known about the glycomic profiling of iPSCs compared with ESCs. For the purpose of clinical applications, the biological characteristics of iPSCs and ESCs before and after differentiation should be identified. Therefore, we performed quantitative glycan analysis to identify the biological difference between iPSCs and ESCs during neuronal differentiation. Our analysis showed that the expression pattern in undifferentiated iPSCs was 2% bisect-type N-glycans and 60% of high-mannose N-glycans, whereas the pattern in ESCs was different from that of iPSCs (1% of bisect-type N-glycans and 75% of high-mannose N-glycans). Although the HILIC–LC/MS method can directly measure the N-glycan released from glycoproteins, its accuracy is insufficient because this method cannot avoid capturing small size peptides which have a hydrophobic property. Our glycoblotting method is theoretically more accurate than HILIC–LC/MS method because this method captures only glycans via a chemoenzymatically specific reaction. Tateno et al. (2011) reported a similar glycoblot pattern in both iPSCs and ESCs using lectin arrays for cellular characterization. Dodla et al. and Alisson-Silva et al. also reported an alteration of glycans during cell differentiation using lectin arrays. However, a limited number of lectins, the proteins which recognize specific glycans, are available with this method and they usually show broad specificity for glycan structures (Alisson-Silva et al., 2014; Dodla et al., 2011). Furthermore, another advantage of our method is that it provides quantitative information. Fujitani et al. performed a comprehensive analysis of the cellular glycomes of human iPSCs and human ESCs, including N-glycans, O-glycans, glycosaminoglycans, glycosphingolipid-associated glycans, and free oligosaccharides. They identified distinct glycoblot patterns for iPSCs and ESCs (Fujitani et al., 2013). In the present study, we report a complete portrait of cellular N-glycan expression during an undifferentiated stage that allows for the identification of characteristic glycotypes. The patterns of cellular glycoforms detected using this precise analysis were distinct, indicating that iPSCs and ESCs are distinguishable by their glycan structures. Although the pre-neuronal differentiation glycoblot patterns were different between iPSCs and ESCs, glycoblot patterns in iPSCs and ESCs became more similar with neuronal differentiation. Moreover, glycoblot patterns in neurons differentiated from iPSCs and ESCs closely resemble that of mature neurons (Fig. 7). During neurogenesis, N-glycan structures appear to contribute to cell–cell and cell–matrix interactions, resulting in targeting behaviors such as cell migration and neurite outgrowth. Therefore, N-glycans are considered to be an important regulatory factor during neuronal differentiation (Gu et al., 2004; Shigeta et al., 2006). The glycoblot profiles of N-glycans revealed that the expression of bisect-type N-glycans was significantly up-regulated in neurons differentiating from iPSCs or ESCs. Bisect-type N-glycans are thought to regulate neurite outgrowth (Shigeta et al., 2006) and neuronal differentiation (Gu et al., 2004). Amano et al. (2010) demonstrated that both mouse embryonic carcinoma cells and ESCs exhibited up-regulation of bisect-type N-glycans during neuronal differentiation. These results suggest that undifferentiated cells, such as embryonic carcinoma cells, iPSCs, and ESCs, exhibit a general pattern of glycan expression during neuronal differentiation.