title: Efficient generation of functional pancreatic β-cells from human induced pluripotent stem cells.
abstract: BACKGROUND
  Insulin-secreting cells have been generated from human embryonic or induced pluripotent stem cells (iPSCs) by mimicking developmental processes. However, these cells do not always secrete glucose-responsive insulin, one of the most important characteristics of pancreatic β-cells. We focused on the importance of endodermal differentiation from human iPSCs in order to obtain functional pancreatic β-cells.
  
  
  METHODS
  A six-stage protocol was established for the differentiation of human iPSCs to pancreatic β-cells using defined culture media without feeders or serum. The effects of CHIR99021, a selective glycogen synthase kinase-3β inhibitor, were examined in the presence of fibroblast growth factor 2, activin, and bone morphogenetic protein 4 (FAB) during definitive endodermal induction by immunostaining for SRY (sex determining region Y)-box 17 (SOX17) and Forkhead box protein A2 (FOXA2). Insulin secretion was compared between the last stage of monolayer culture and spheroid culture conditions. Cultured cells were transplanted under kidney capsules of streptozotocin-diabetic non-obese diabetic-severe combined immunodeficiency mice, and blood glucose levels were measured once a week. Immunohistochemical analyses were performed 4 and 12 weeks after transplantation.
  
  
  RESULTS
  Addition of CHIR99021 (3 μmol/L) in the presence of FAB for 2 days improved endodermal cell viability, maintaining the high SOX17-positive rate. Spheroid formation after the endocrine progenitor stage showed more efficient insulin secretion than did monolayer culture. After cell transplantation, diabetic mice had lower blood glucose levels, and islet-like structures were detected in vivo.
  
  
  CONCLUSIONS
  Functional pancreatic β-cells were generated from human iPSCs. Induction of definitive endoderm and spheroid formation may be key steps for producing these cells.
authors.affiliation: Department of Regenerative Medicine, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan.
authors.forename: Shigeharu G
authors.initials: SG
authors.lastname: Yabe
authors.affiliation: Department of Regenerative Medicine, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan.
authors.forename: Satsuki
authors.initials: S
authors.lastname: Fukuda
authors.affiliation: Department of Regenerative Medicine, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan.
authors.forename: Fujie
authors.initials: F
authors.lastname: Takeda
authors.affiliation: Department of Regenerative Medicine, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan.
authors.forename: Kiyoko
authors.initials: K
authors.lastname: Nashiro
authors.affiliation: Pancreatic Islet Transplantation Project, Department of Diabetes Research Center, National Center for Global Health and Medicine, Tokyo, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Shimoda
authors.affiliation: Department of Regenerative Medicine, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Okochi
mesh_terms: D028341:Activins / Q000494:pharmacology; D000818:Animals; D055415:Bone Morphogenetic Protein 4 / Q000494:pharmacology; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics*; D002478:Cells, Cultured; D003921:Diabetes Mellitus, Experimental / Q000235:genetics / Q000378:metabolism / Q000628:therapy; D004707:Endoderm / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D051542:Hepatocyte Nuclear Factor 3-beta / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D057026:Induced Pluripotent Stem Cells / Q000187:drug effects / Q000378:metabolism* / Q000637:transplantation; D007328:Insulin / Q000235:genetics / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055760:SOXF Transcription Factors / Q000235:genetics / Q000378:metabolism; D018874:Spheroids, Cellular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D033581:Stem Cell Transplantation / Q000379:methods; D014183:Transplantation, Heterologous
chemical_list: D055415:Bone Morphogenetic Protein 4; C473711:Chir 99021; D007328:Insulin; D011725:Pyridines; D011743:Pyrimidines; D055760:SOXF Transcription Factors; C422405:activin A; D016222:Fibroblast Growth Factor 2; D028341:Activins; D051542:Hepatocyte Nuclear Factor 3-beta
keywords: diabetes; induced pluripotent stem cells; pancreatic β-cells