Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-07
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • 2024-04
  • 2024-05

    • TGF type I receptor inhibitors were also found to induce

    2018-11-06

    TGF-β type I receptor inhibitors were also found to induce differentiation of insulin-producing cells. Among the TGF-β type I receptor inhibitors that we tested, Alk5 inhibitor II showed unique effects on the differentiation of pancreatic endocrine cells. Only Alk5 inhibitor II significantly increased the expression of both INS and GCG, unlike the other TGF-β type I receptor inhibitors (Fig. 4B and data not shown). In the kinase profiling assay, Alk5 inhibitor II inhibited various kinases at the concentration used in this study (data not shown), suggesting that it might regulate the differentiation of pancreatic endocrine 5z by inhibiting various other signals in addition to those of TGF-β. The mechanisms involved in differentiation of pancreatic progenitor cells into each type of hormone-producing cell are largely unknown, so elucidating the mechanism by which Alk5 inhibitor II acts would be helpful for understanding the process of pancreatic endocrine cell differentiation. Rezania et al. reported that insulin and glucagon double-positive cells were induced by treatment with Alk5 inhibitor II and that most of these cells finally differentiated into glucagon-producing alpha cells (Rezania et al.). When pancreatic progenitor cells were treated with Alk5 inhibitor II alone in our culture system, the percentage of both glucagon-positive cells and glucagon and insulin double-positive cells was relatively high (Fig. 4D), a finding that was consistent with their results. However, when the cells were treated with four compounds (forskolin, dexamethasone, Alk5 inhibitor II, and nicotinamide), induction of these cells was much lower and most of the insulin-positive cells that we obtained were also positive for PDX1(Figs. 4D, 5A, and Fig. S5), which is not expressed in glucagon-producing cells in the human fetal pancreas (Lyttle et al., 2008). Moreover, the number of insulin-positive (glucagon-negative) cells was much larger than that of glucagon-positive cells, even after additional four weeks of culture (data not shown). These results indicate that cells were more likely to undergo differentiation into pancreatic β-cells by treatment with the four compounds (forskolin, dexamethasone, Alk5 inhibitor II, and nicotinamide) in our culture system. It is noteworthy that multiple hiPS cell lines showed differentiation into insulin-producing cells by our protocol. The hiPS cell lines that we utilized in this study were generated from the fibroblasts of various individuals, including a neonatal male and a 73-year-old woman (Takahashi et al., 2007; Nakagawa et al., 2008; Takahashi et al., 2009). Thus, our results suggest that this is a robust protocol and that optimization for individual cell lines might not be required. In another report, the rate of differentiation into insulin-producing cells varied among different hES cell lines (D\'Amour et al., 2006). Since stable differentiation from various hiPS cell lines is necessary for clinical application, our protocol could be beneficial for development of cell therapy in the future. In conclusion, we developed a simple method for promoting the differentiation of hiPS cells into insulin-producing cells. With this method, all of the hiPS cell lines that we tested showed efficient differentiation into insulin-producing cells, suggesting that our protocol achieves efficient and reproducible generation of insulin-producing cells in vitro. This protocol has the potential to contribute to drug discovery and cell therapy for diabetes.
    Materials and methods
    Acknowledgments
    Introduction Hypoxic preconditioning has been investigated as a neuroprotective strategy although the exact details are not well understood. Known mechanisms in this pathway involve the upregulation of the hypoxia-inducible factor (HIF-1α) and erythropoietin (Epo) (Kumral et al., 2003; Grimm et al., 2005; Liu et al., 2005; Grimm et al., 2006), both of which have been shown to be neuroprotective agents (Digicaylioglu and Lipton, 2001; Buemi et al., 2002; Juul, 2002; Yu et al., 2002; Kumral et al., 2003; Hasselblatt et al., 2006). The hypoxia signaling cascade appears to be vital in preconditioning, and several studies have demonstrated the importance of HIF-1α. For example, inactivation of HIF-1α increased brain injury (Baranova et al., 2007), and blocking HIF-1α activation with low-dose cadmium attenuated myocardial hypoxic preconditioning (Belaidi et al., 2008). Additionally, other agents such as insulin growth factor 1 (IGF-1) and cobalt chloride have been shown to activate or increase HIF-1α and lead to preconditioning (Wang et al., 2004; Xi et al., 2004). Heat shock protein 27 (Hsp27) also increases HIF-1α and causes protection against retinal ischemia (Whitlock et al., 2005). In vitro studies in C6 glioma cells demonstrated that increased HIF-1α decreased the insults caused by 3-nitroproprionic acid (Yang and Levison, 2006).