Objective To investigate whether pancreatic progenitors and islets differentiated from human embryonic stem cells(hESCs) could correct hyperglycemia in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice.Methods We obtained pancreatic progenitors and islets derived from hES cells line YT1 according to the optimized four-stage differentiation protocol.Stage 1:definitive endoderm formation; Stage 2:pan creatic specialization; Stage 3:amplification of pancreatic progenitors,Stage 4:maturation of pancreatic islets.To observe the morphological changes of each stage and immunofluorescent expression of pancreatic and duodenal homeobox gene(PDX-1),glucagon,insulin,C-peptide,glucose transporter-2(Glut-2).The differentiated cells from stage 3 and stage 4 were then transplanted into one of the epididymal fat pads(EFP) of NOD/SCID mice.The survival and function of the graft were measured by immunohistochemistry and blood glucose monitor.Results The stage 4-differentiated pancreatic islets expressed mature β cell-specific markers such as glucagon,insulin and Glut 2,and even PDX-1 and C-peptide-double-positive.The stage 4-pancreatic islets had nearly 17.1％ insulin-positive cells as assayed by flow cytometry analysis.Differentiated pancreatic islets released insulin/C-peptide in response to glucose stimulation.After implantation into EFP of NOD/SCID mice,hES cell-derived human pancreatic progenitors and islets corrected hyperglycemia for at least 12 weeks.Conclusions Pancreatic progenitors and islets differentiated from hESCs can correct hyperglycemia in NOD/SCID mice.

Objective To investigate whether pancreatic progenitors differentiated from human embryonic stem(hES) cells could correct hyperglycemia in non-obese diabetic(NOD)/severe combined immunodeficient(SCID) mice or not.Methods Pancreatic islets derived from hES cells line YT1 according to the optimized four-stage differentiation protocol in a chemical-defined culture system were observed.In the first stage,activin A and wortmannin were utilized to induce definitive endoderm formation.In the second stage,the differentiated endoderm cells were treated with all-trans retinoic acid(RA),NOGGIN and basic fibroblast growth factor(bFGF) to induce pancreatic specialization.In the third stage,pancreatic progenitors were induced by epidermal growth factor(EGF)-regulated expansion.Finally,in the fourth stage,a cocktail of factors was utilized to induce mature insulin-producing cells.The differentiated human pancreatic islet cells of(3-5)×106 were then transplanted into one of the epididymal fat pads(EFP) of NOD/SCID mice.Graft survival and function were measured by blood glucose levels.Results The differentiated hES cells obtained by the four-stage approach comprised nearly 17.1% insulin-positive cells and 3.8% C-peptide positive as assayed by flow cytometry analysis,which released insulin/C-peptide in response to glucose stimuli in a manner comparable to that of adult human islets.Most of these insulin-producing cells co-expressed mature β cell-specific markers such as PDX1,insulin,C-peptide and glucagon.After implantation into EFP of NOD/SCID mice,hES cell-derived pancreatic islets corrected hyperglycemia for an least eight weeks.Conclusion Human terminal differentiation pancreatic islet cells can correct hyperglycemia in NOD/SCID mice.