br Source of funding This work

Source of funding This work was supported by research funding from The Ministry of Education, Culture, Sports, Science and Technology Grant-in-Aid for Scientific Research (C) (22591555) and the Riken Center for Developmental Biology Collaborative Research Fund, Kobe (08001475).
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Acknowledgments
Introduction Diabetes is caused by an absolute or relative insufficiency of insulin which is secreted from pancreatic β-cells, resulting in impaired glucose metabolism in the entire body (Mathis and Vence, 2001; Donath and Halban, 2004). The supplementation of β-cell function is an effective therapeutic strategy, but the insufficient cell supply is a major obstacle to this intervention (Matsumoto, 2011). Therefore, vigorous efforts have been made to develop a stable source of pancreatic adenosine receptor antagonist for clinical use. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) are attractive cell sources due to their potential for unlimited proliferation and differentiation (Dominguez-Bendala et al., 2011; McCall et al., 2010). Indeed, a number of groups have generated immature pancreatic β cell-like cells, which are referred to as insulin-producing or insulin-secreting cells, from hESCs/iPSCs in vitro (D\'Amour et al., 2006; Jiang et al., 2007a,b; Zhang et al., 2009; Nostro et al., 2011; Kroon et al., 2008; Mfopou et al., 2010; Shim et al., 2007; Cai et al., 2010; Kelly et al., 2011; Xu et al., 2011; Kunisada et al., 2012; Rezania et al., 2011, 2012, 2013; Schulz et al., 2012; Bruin et al., 2013; Sui et al., 2013). Despite the difficulties in generating genuine β-cells in vitro, mature insulin-producing cells with the potential for glucose responsiveness have been obtained following the implantation of hESC-derived pancreatic progenitors into immunodeficient mice (D\'Amour et al., 2006; Kroon et al., 2008; Shim et al., 2007; Kelly et al., 2011; Rezania et al., 2012, 2013; Bruin et al., 2013). Therefore, implantation of pancreatic progenitors, instead of β-cells, is also considered to be a therapeutic option for diabetes. Previous reports have described the generation of pancreatic lineage cells, such as pancreatic progenitors and insulin-producing cells, from hESCs/iPSCs in vitro, using stepwise differentiation protocols that recapitulate pancreatic development (D\'Amour et al., 2006; Jiang et al., 2007a,b; Zhang et al., 2009; Nostro et al., 2011; Kroon et al., 2008; Mfopou et al., 2010; Shim et al., 2007; Cai et al., 2010; Kelly et al., 2011; Xu et al., 2011; Kunisada et al., 2012; Rezania et al., 2011, 2012, 2013; Schulz et al., 2012; Bruin et al., 2013; Sui et al., 2013). However, the quantitative differences in the differentiation efficiency among hESC/iPSC lines make it difficult to obtain the stable production of pancreatic lineage cells with sufficient purity for use in the clinical setting (Osafune et al., 2008). One of the solutions for this is the establishment of stable differentiation methods that can be more broadly applied to multiple hESC/iPSC lines, based on a complete understanding of the mechanisms of pancreatic organogenesis. The major components of the pancreas are exocrine, duct and endocrine cells, which originate from the endoderm layer of early embryos marked by the co-expression of transcription factors Sox17 and FoxA2 (Jennings et al., 2013). The endodermal layer extends and folds to form the primitive gut tube expressing Hnf1β and Hnf4α, which has potential to differentiate into respiratory apparatus, digestive tract and endocrine organs, including the pancreas. Distinct pancreatic organogenesis morphologically starts at the posterior foregut area in the primitive gut tube following the expression of a transcription factor, Pdx1 (Jorgensen et al., 2007; Jensen, 2004). Two parts of the Pdx1+ gut tube thicken to form the dorsal and ventral pancreatic buds. It should be noted that the early pancreatic epithelia formed by the stratification of the single cell layer of the gut tube and its protrusion into the surrounding mesenchyme exist as a three-dimensional highly-dense aggregate expressing additional transcription factors, Ptf1a and Nkx6.1, during the process of bud formation (Hald et al., 2008; Ahnfelt-Ronne et al., 2012; Kawaguchi et al., 2002; Villasenor et al., 2010). After that, microlumens appear and fuse to form epithelial structures with a branched tubular network within the aggregated pancreatic buds. Some epithelia differentiate into hormone-producing endocrine lineages, such as insulin-secreting β-cells and glucagon-secreting α-cells, and migrate into the mesenchyme to form pancreatic islets. Therefore, during the long process of islet formation, cells experience dynamic structural changes.