A cDNA that encodes a glutathione-S-transferase belonging to sigma class (GSTS1) from the silkworm, Bombyx mori was cloned by reverse transcriptase-polymerase chain reaction and sequenced. The deduced amino acid sequence revealed 66%, 48% and 41% identity to sigma-class GSTs from Manduca sexta, Blattella germanica and Anopheles gambiae, respectively. The GSTS1 was also estimated to be close to those GSTs in a phylogenetic tree. GSTS1 mRNA was widely distributed in various tissues. The recombinant GST (rGSTS1) was functionally overexpressed in Escherichia coli in a soluble form, purified to homogeneity and characterized. The pH-optimum of rGSTS1 was around pH 8. The rGSTS1 retained more than 75% of its original activity after incubation at pH 4-9. Incubation for 30 min at temperatures below 40°C did not affect its activity. rGSTS1 was able to catalyse the reaction of glutathione with 1-chloro-2,4-dinitrobenzene, the universal substrate for GST, as well as with 4-hydroxynonenal, the product of lipid peroxidation.

Aims/hypothesis The generation of induced pluripotent stem cells (iPSCs) provides a promising possibility for type 1 diabetes therapy. However, the generation of insulinproducing cells from iPSCs and evaluation of their efficacy and safety should be achieved in large animals before clinically applying iPSC-derived cells in humans. Here we try to generate insulin-producing cells from rhesus monkey (RM) iPSCs. Methods Based on the knowledge of embryonic pancreatic development, we developed a four-stage protocol to generate insulin-producing cells from RM iPSCs. We established a quantitative method using flow cytometry to analyse the differentiation efficiency. In addition, to evaluate the differentiation competence and function of RM iPSC-derived cells, transplantation of stage 3 and 4 cells into immunodeficient mice was performed. Results RM iPSCs were sequentially induced to definitive endoderm (DE), pancreatic progenitors (PP), endocrine precursors (EP) and insulin-producing cells. PDX1 + PP cells were obtained efficiently from RM iPSCs (over 85% efficiency). The TGF-β inhibitor SB431542 promoted the generation of NGN3 + EP cells, which can generate insulin-producing cells in vivo upon transplantation. Finally, after this fourstage differentiation in vitro, insulin-producing cells that could secrete insulin in response to glucose stimulation were obtained. When transplanted into mouse models for diabetes, these insulin-producing cells could decrease blood glucose levels in approximately 50% of the mice.Conclusions/interpretation We demonstrate for the first time that RM iPSCs can be differentiated into functional insulin-producing cells, which will provide the basis for investigating the efficacy and safety of autologous iPSCderived insulin-producing cells in a rhesus monkey model for type 1 diabetes therapy.