br Introduction Previously it was
Introduction Previously, it was thought that the fully developed mammalian central nervous system (CNS) lacks significant regenerative capacity (Ramon, 1928). However, the recent discovery of neural progenitor cells, including neural stem alcohol dehydrogenase inhibitor (NSCs), has shown a potential for overcoming this limitation in repairing the damaged CNS (Reynolds and Weiss, 1992; Taupin and Gage, 2002; van Praag et al., 2002). The morphology and function of NSCs change drastically during the course of neuronal differentiation from NSCs to neurons (Nakayama and Inoue, 2006; Nakayama et al., 2004). Despite a large number of studies aimed at clarifying the mechanisms of neuronal differentiation, many important questions remain unanswered. Induced pluripotent stem cells (iPSCs) generated from somatic cells via direct molecular reprogramming are capable of unlimited proliferation and differentiation into multiple types of mature cells (Takahashi et al., 2007; Takahashi and Yamanaka, 2006). Mouse and human iPSCs are similar to their respective embryonic stem cells (ESCs) in terms of morphology, proliferation, gene expression, and teratoma formation (Takahashi et al., 2007). It is believed that these stem cells hold tremendous promise with regard to applications in regenerative medicine. However, current methods for assessing differential properties are limited in scope and a comprehensive method that directly targets stem cells is lacking. Development of such methodologies is an essential prerequisite for the clinical application of iPSCs and ESCs. Although a number of functional genomic and proteomic studies of cellular differentiation have been conducted in recent years (Gonnet et al., 2008; Kuramitsu and Nakamura, 2006; Watkins et al., 2008), no reliable molecular markers have emerged that can be used to determine the quality and differentiation status of iPSCs and ESCs. Glycans attached to proteins (glycoproteins) and sphingolipids (glycosphingolipids) are located at the outermost surface of the cell. Significant alterations in the cellular glycome occur during development and cellular differentiation (Amano et al., 2010; Kannagi et al., 1983; Lanctot et al., 2007; Lau et al., 2007), suggesting that glycans could serve as novel cellular differentiation biomarkers. Unfortunately, technical difficulties have hampered efforts to determine the total glycan complement of mammalian cells and have limited the assessment of cell type-specific glycoforms. To overcome these methodological issues, we developed a glycoblotting method based on a PCR-like technology that allows for rapid and quantitative glycan analysis (Amano et al., 2010). The majority of glycoproteins are classified into 2 groups: those attached to an asparagine residue through a nitrogen atom (N-glycans) and those attached to a serine or a threonine residue (O-glycans). Cellular responses to signals that stimulate growth or cell-cycle arrest depend heavily on the total number of N-glycan molecules on the surface of the cell and the degree of branching of cell surface glycoproteins (Lau et al., 2007). Of these 2 types of glycans, modifications of N-glycans on proteins are thought to contribute to embryogenesis and organogenesis. However, a full portrait of glycome diversity and the effect of cellular glycoform structural variations on individual cell stages during proliferation and differentiation remain unclear. Therefore, we quantitatively monitored dynamic glycoform changes during neuronal differentiation of mouse iPSCs and ESCs by using this glycoblotting method. The hypothesis of this study is that alterations in N-glycans occur during neuronal differentiation of mouse ESC and iPSC-derived NSCs. The objectives of this study are to identify alterations in N-glycans and the gene expression of an associated N-glycan biosynthesis enzyme during the course of neuronal differentiation in mice. Additionally, we compared the glycoform pattern in neuronal cells differentiated from iPSCs or ESCs.