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  • In summary we demonstrate that GRM functions as

    2018-10-24

    In summary, we demonstrate that GRM7 functions as a modulator of neurogenesis. We identify CREB, YAP, and CYCLIND1 as the downstream targets of GRM7. The phosphorylation of CREB at Ser133 and the expression of YAP and CyclinD1 are modulated by GRM7, thereby regulating neurogenesis. Future behavioral studies will facilitate a deeper understanding of the relationship between GRM7 and developmental brain disorders.
    Experimental Procedures
    Author Contributions
    Acknowledgments
    Introduction Human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) (Thomson et al., 1998) and human induced pluripotent stem cells (hiPSCs) (Takahashi et al., 2007), offer immense potential as cell sources for cell-based therapies because of their capacity for unlimited self-renewal and pluripotent differentiation. In particular, hiPSCs are creating great expectations not only for regenerative medicine, but also for disease modeling and drug development, as they can be generated from various adult somatic cells simply by introducing reprogramming factors. Enormous efforts have been undertaken to establish hPSC-based therapies for a variety of degenerative diseases (Garber, 2013). Recently, the first in-human clinical trial using hiPSC-derived retinal pigment epithelium was conducted by RIKEN Center for Developmental Biology in Kobe to treat the wet form of age-related macular degeneration (Kamao et al., 2014). However, although the clinical and industrial application of hPSC-based cell therapy is becoming an increasingly realistic prospect, a major safety concern still exists, as residual hPSCs in differentiated cell populations could form tumors in recipients (Ben-David and Benvenisty, 2011; Goldring et al., 2011; Lee et al., 2013a). Over the past several years, the tumorigenicity risks of hPSCs have been highlighted in a number of animal studies (Hentze et al., 2009; Kawai et al., 2010; Lee et al., 2009; Roy et al., 2006; Yamashita et al., 2011). As few as 100 hPSCs have been reported to be sufficient to produce a teratoma (Gropp et al., 2012; Hentze et al., 2009). Therefore, complete elimination of hPSCs from the final cell products without compromising their viability, safety, efficacy, and functional properties is a prerequisite for clinical application of hPSC-based therapy. It is also important to remove residual hPSCs from hPSC-derived cells to establish disease models. Several strategies to remove residual hPSCs from differentiated cell cultures have been reported, including the introduction of suicide genes into hPSCs (Schuldiner et al., 2003), selective killing using cytotoxic Lomustine (Ben-David et al., 2013b; Choo et al., 2008; Tan et al., 2009) and chemical inhibitors (Ben-David et al., 2013a; Lee et al., 2013b; Richards et al., 2014; Vazquez-Martin et al., 2012), cell sorting using hPSC-specific antibodies (Ben-David et al., 2013b; Tang et al., 2011) and lectins (Wang et al., 2011), and glucose deprivation in the cell culture medium (Tohyama et al., 2013). However, all of these methods have some limitations in terms of specificity, throughput, efficacy, and safety. The development of alternative strategies based on different mechanisms therefore is warranted. Previously, we performed comprehensive glycome analysis of a large number of hPSCs (114 types of hiPSCs and nine types of hESCs) using high-density lectin microarrays. We found that a lectin designated rBC2LCN (recombinant N-terminal domain of BC2L-C lectin derived from Burkholderia cenocepacia) binds to various types of hiPSCs and hESCs, but not to differentiated somatic cells (Tateno et al., 2011). rBC2LCN is a useful hPSC probe, which strongly stains formaldehyde-fixed hPSCs at the cell membrane (Onuma et al., 2013). In addition, it allows live staining of hPSCs after supplementation of the culture medium, with no visible signs of toxicity. The staining is specific to undifferentiated cells, and rapidly diminishes upon their differentiation. Detailed specificity analysis revealed that rBC2LCN binds to the defined glycan structure, Fucα1-2Galβ1-3GlcNAc/GalNAc (Sulák et al., 2010; Tateno et al., 2011). Among the N- and O-glycans isolated from 201B7 hiPSCs, a core2-type O-glycan, Fucα1-2Galβ1-3(Galβ1-4GlcNAcβ1-6)GalNAc, was found to contain the above glycan structure (Hasehira et al., 2012). rBC2LCN exhibited a significant affinity (Ka of 2.5 × 104 M−1) to this core2-type O-glycan, demonstrating that it is a glycan ligand of rBC2LCN (Tateno et al., 2013). Furthermore, podocalyxin, a type1 transmembrane protein, was identified as a predominant glycoprotein ligand of rBC2LCN (Tateno et al., 2013). Podocalyxin is a hyperglycosylated sialomucin containing a mucin domain with >100 putative O-glycosylation sites, five potential N-linked glycosylation sites, and three putative glycosaminoglycan sites (Kershaw et al., 1997). Due to its heavily glycosylated nature, the apparent molecular weight of podocalyxin expressed on hPSCs is >200 kDa, despite the calculated molecular weight of 55 kDa (Tateno et al., 2013). Although the binding affinity of rBC2LCN to the core2-type O-glycan is relatively low, that to 201B7 hiPSCs is as high as antibody (Ka = 5 × 108 M−1), presumably due to the high-density display of the glycan ligand on hPSCs, the so-called “glycoside cluster effect” (Dam and Brewer, 2010; Dam et al., 2009). Most recently, the hyperglycosylated podocalyxin was found to be secreted into cell culture media. Taking advantage of this phenomenon, a noninvasive and quantitative detection system of tumorigenic hPSCs in transplanting cells using cell culture media was developed (Tateno et al., 2014).