title: Suppression of NANOG induces efficient differentiation of human embryonic stem cells to pancreatic endoderm.
abstract: OBJECTIVE
  A challenge in using human embryonic stem cells (hESCs) as the source of surrogate β cells is the establishment of methods that could effectively direct their differentiation into functional β cells. The aim of this study was to assess the effect of NANOG gene suppression in differentiating hESCs as a mean of increasing the efficiency with which endoderm-derived pancreatic cells could be generated.
  
  
  METHODS
  A homogenous cell population with stable suppression of NANOG was generated in hESC ENVY line using plasmid-based siRNA approach. Pancreatic differentiation was undertaken according to the ontology-based in vitro selection protocol and followed by transplantation into immunodeficiency mice to mature in vivo.
  
  
  RESULTS
  We observed up-regulation of definitive endoderm genes, which expand the role of NANOG in blocking definitive endoderm differentiation. The ontology-based differentiation protocol resulted in increased expression of markers essential for pancreatic epithelium development. Transplantation of these cells further revealed a homogenous pancreatic exocrine-like morphology that stained positively for amylase.
  
  
  CONCLUSIONS
  The suppression of NANOG displayed an effective differentiation toward endoderm and pancreatic progenitors. Investigation of the factors required for endocrine formation combined with a prolonged in vivo culturing could be further used to increase the ratio of endocrine-exocrine cells fate.
authors.affiliation: Diabetes Transplant Unit, Prince of Wales Hospital & The University of New South Wales, Australia. khun.lie@unsw.edu.au
authors.forename: Khun-Hong
authors.initials: KH
authors.lastname: Lie
authors.forename: Bernard E
authors.initials: BE
authors.lastname: Tuch
authors.forename: Kuldip S
authors.initials: KS
authors.lastname: Sidhu
mesh_terms: D000681:Amylases / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D004707:Endoderm / Q000166:cytology* / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D008858:Microscopy, Phase-Contrast; D000071317:Nanog Homeobox Protein; D010179:Pancreas / Q000166:cytology* / Q000378:metabolism; D046790:Pancreas, Exocrine / Q000166:cytology / Q000378:metabolism; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D033581:Stem Cell Transplantation / Q000379:methods; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism
chemical_list: D018398:Homeodomain Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D000681:Amylases