br Results br Discussion As the first step

Results
Discussion As the first step of our method, hiPS cells underwent differentiation into definitive endoderm by treatment with activin A and the GSK3β inhibitor CHIR99021. In other methods, activin A and Wnt 3a have mainly been used to induce definitive endoderm (D\'Amour et al., 2006; Kroon et al., 2008; Maehr et al., 2009; Chen et al., 2009; Ameri et al.; Johannesson et al., 2009). However, we found that treatment with activin A plus CHIR99021 induced SOX17 and FOXA2 double-positive definitive endoderm more efficiently compared with activin A plus Wnt 3a. We confirmed that CHIR99021 was a highly selective inhibitor of GSK3β and GSK3α by the kinase profiling assay (KinaseAdvisor™, Caliper LifeSciences) (data not shown), suggesting that its influence on other signaling processes would be very small. In consideration of efficiency, stability, and cost, using CHIR99021 instead of Wnt 3a is preferable for inducing differentiation into definitive endoderm. Next, we induced differentiation of definitive endoderm into pancreatic progenitor cells. A large number of factors, including Cyclopamine-KAAD, (−)-indolactam V, Noggin, retinoic acid, FGF2, FGF4, FGF10, and EGF, have been reported to induce the differentiation of pancreatic progenitor cells (D\'Amour et al., 2006; Kroon et al., 2008; Jiang et al., 2007; Chen et al., 2009; Cai et al.; Ameri et al.; Johannesson et al., 2009). Among these factors, we found combined treatment with Noggin and retinoic adenylate cyclase was effective for inducing PDX1-positive cells in our culture condition. We also showed that treatment with the BMP receptor inhibitor dorsomorphin and retinoic acid could induce the differentiation of PDX1-positive cells. This finding is consistent with several recent reports that Noggin can be replaced by dorsomorphin in the induction of ES cell differentiation (Hao et al., 2008; Wada et al., 2009; Nostro et al., 2011). Because of its superior stability and lower cost, dorsomorphin seemed to be a better choice than Noggin for induction of PDX1-positive cells. Furthermore, we found that inhibitors of the TGF-β type I receptor were effective for inducing pancreatic differentiation in combination with retinoic acid and dorsomorphin. This is consistent with a recent report that PDX1 expression was induced by treatment of mouse embryos with SB431542 at the 5–6 somite stage (Wandzioch and Zaret, 2009). Moreover, while we were preparing this manuscript, Rezania et al. and Nostro et al. reported that treatment with TGF-β type I receptor inhibitors following pancreatic specification induced the differentiation into pancreatic endocrine cells from human ES cells (Nostro et al., 2011; Rezania et al.). Our results were also consistent with their reports. Thus, TGF-β signaling might have a negative influence on differentiation into pancreatic progenitor cells. As the next step, we investigated conditions that allowed the efficient generation of insulin-producing cells from PDX1-positive cells. Various compounds, including forskolin, dbcAMP, IBMX, TGF-β type I receptor inhibitors, and dexamethasone, have been found to increase the number of insulin-producing cells. Since forskolin and IBMX are known to increase the intracellular cAMP level and dbcAMP is a cell-permeable cAMP analog, it is suggested that the cellular cAMP level is one of the key factors regulating the differentiation of insulin-producing cells. However, we found that long-term exposure (>20days) to forskolin alone decreased the number of insulin-producing cells (data not shown). Thus, a high level of cAMP might be needed in priming for differentiation, but excess cAMP might have a negative effect on the survival of insulin-producing cells. Interestingly, when cells were treated with dexamethasone in addition to forskolin, the number of insulin-producing cells remained stable for a long period. The precise mechanism remains to be analyzed, but this finding indicates that glucocorticoid signaling promotes the survival of insulin-producing cells.