The differentiated cells were cultured in RPMI1640 supplemented with 0.5% FBS, 0.5% insulin/transferrin/selenium (ITS), 0.5B27, 2 M retinoic acidity (RA) (Sigma, St. function were assessed using immunohistochemistry, and measuring serum human C-peptide and blood glucose levels. Results The pancreatic IPCs were generated by the four-stage differentiation protocol using hESCs. About 17.1% of differentiated cells expressed insulin, as determined by flow cytometry. These cells secreted insulin/C-peptide following glucose stimulation, similarly to adult human islets. Most of these IPCs co-expressed mature cell-specific markers, including human C-peptide, GLUT2, PDX1, insulin, and glucagon. After implantation into the epididymal excess fat pad of SCID/NOD mice, the hESC-derived pancreatic IPCs corrected hyperglycemia for 8 weeks. None of the animals transplanted with pancreatic IPCs developed tumors during the time. The mean survival of recipients was increased by implanted IPCs as compared to implanted undifferentiated hESCs ( em P /em 0.0001). Conclusions The results of this study confirmed that human terminally differentiated pancreatic IPCs derived from hESCs can correct hyperglycemia in SCID/NOD mice for 8 weeks. Introduction The development of a cellular therapy for diabetes requires a renewable source of human insulin-secreting cells that respond to glucose in a physiologic manner. Mature islet transplantation has been proposed as a encouraging treatment for type 1 diabetes , Rucaparib . However, an acute shortage of deceased organ donors currently limits the wider application of islet transplantation. One approach to overcome the limited supply of donor pancreases is usually to generate Rucaparib IPCs from stem cells with high proliferative and differentiating potential . hESCs have the potential to differentiate into specialized cells of all three main germ-layers, including pancreatic IPCs Rucaparib , . hESCs symbolize a potentially unlimited source of transplantable islet cells for treating diabetes . For this reason, systematic and mechanistic studies are required to examine the potential for using hESCs as a stem cell-based therapy for type 1 diabetes. Several groups have reported stepwise protocols for mimicking the development of the pancreas in vivo. D’Amour Rabbit polyclonal to CDK4 et al  reported a five-stage protocol for differentiating hESCs Rucaparib into pancreatic hormone-expressing endocrine cells that secreted insulin in response to numerous secretagogues but not to glucose in vitro. Zhang et al  reported a four-stage protocol for differentiating hESCs into mature IPCs that secreted insulin/C-peptide in response to glucose stimulation. After comparing the different protocols, we chose a four-stage protocol for inducing the differentiation of hESCs into IPCs, and transplanted the cells into SCID/NOD mice to assess graft survival and function by performing immunohistochemistry, and measuring serum human C-peptide levels and blood glucose levels. We found that these terminally differentiated cells were morphologically and functionally much like pancreatic islets, and guarded mice against streptozotocin (STZ)-induced hyperglycemia. Methods hESC culture and differentiation This study was approved by Ethics Committee of The Medical College of Qingdao University or college, China. The hESC lines YT1 and YT2  were derived and characterized at our institute. The hESCs were cultured in Dulbecco’s altered Eagle’s medium (DMEM)/F12 supplemented with 20% KnockOut serum replacement (KSR) Rucaparib and 4 ng/mL of basic fibroblast growth factor (bFGF) on mouse embryonic fibroblast feeders. Colonies of hESCs were digested with 10 mg/mL collagenase IV into small clumps for differentiation. The hESC clumps were replated on Matrigel (BD Biosciences, Franklin Lakes, NJ, USA; 150)-coated dishes to provide protection of 60%. The cells were incubated with RPMI1640 made up of 0.2% fetal bovine serum (FBS), 0.5N2 and 0.5B27 supplemented with 100 ng/mL activin A (R&D Systems, Minneapolis, MN, USA) and 1 M wortmannin for 4 days. The differentiated cells were cultured in RPMI1640 supplemented with 0.5% FBS, 0.5% insulin/transferrin/selenium (ITS), 0.5B27, 2 M retinoic acid (RA) (Sigma, St. Louis, MO, USA), 20 ng/ml fibroblast growth factor-7 (FGF-7), and 50 ng/mL Noggin for 4.
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