title: INS(GFP/w) human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells.
abstract: OBJECTIVE
  We aimed to generate human embryonic stem cell (hESC) reporter lines that would facilitate the characterisation of insulin-producing (INS⁺) cells derived in vitro.
  
  
  METHODS
  Homologous recombination was used to insert sequences encoding green fluorescent protein (GFP) into the INS locus, to create reporter cell lines enabling the prospective isolation of viable INS⁺ cells.
  
  
  RESULTS
  Differentiation of INS(GFP/w) hESCs using published protocols demonstrated that all GFP⁺ cells co-produced insulin, confirming the fidelity of the reporter gene. INS-GFP⁺ cells often co-produced glucagon and somatostatin, confirming conclusions from previous studies that early hESC-derived insulin-producing cells were polyhormonal. INS(GFP/w) hESCs were used to develop a 96-well format spin embryoid body (EB) differentiation protocol that used the recombinant protein-based, fully defined medium, APEL. Like INS-GFP⁺ cells generated with other methods, those derived using the spin EB protocol expressed a suite of pancreatic-related transcription factor genes including ISL1, PAX6 and NKX2.2. However, in contrast with previous methods, the spin EB protocol yielded INS-GFP⁺ cells that also co-expressed the beta cell transcription factor gene, NKX6.1, and comprised a substantial proportion of monohormonal INS⁺ cells.
  
  
  CONCLUSIONS
  INS(GFP/w) hESCs are a valuable tool for investigating the nature of early INS⁺ progenitors in beta cell ontogeny and will facilitate the development of novel protocols for generating INS⁺ cells from differentiating hESCs.
authors: Monash Immunology and Stem Cell Laboratories, Level 3, Building 75, STRIP1, West Ring Road, Monash University, Clayton, Victoria 3800, Australia.
authors: S J
authors: SJ
authors: Micallef
authors: X
authors: X
authors: Li
authors: J V
authors: JV
authors: Schiesser
authors: C E
authors: CE
authors: Hirst
authors: Q C
authors: QC
authors: Yu
authors: S M
authors: SM
authors: Lim
authors: M C
authors: MC
authors: Nostro
authors: D A
authors: DA
authors: Elliott
authors: F
authors: F
authors: Sarangi
authors: L C
authors: LC
authors: Harrison
authors: G
authors: G
authors: Keller
authors: A G
authors: AG
authors: Elefanty
authors: E G
authors: EG
authors: Stanley
mesh_terms: D002454:Cell Differentiation; D002460:Cell Line; D002999:Clone Cells; D003922:Diabetes Mellitus, Type 1 / Q000628:therapy; D058732:Embryoid Bodies / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism* / Q000637:transplantation; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000235:genetics / Q000378:metabolism*; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000378:metabolism* / Q000637:transplantation; D060850:LIM-Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins; D020411:Oligonucleotide Array Sequence Analysis; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D029961:Zebrafish Proteins
chemical_list: D005136:Eye Proteins; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins; D007328:Insulin; D060850:LIM-Homeodomain Proteins; C000715631:NKX2-2 protein, human; C105201:NKX6-1 protein, human; D009687:Nuclear Proteins; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D051761:Paired Box Transcription Factors; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D029961:Zebrafish Proteins; C485184:enhanced green fluorescent protein; C063843:insulin gene enhancer binding protein Isl-1; C000715630:nkx2.2b protein, zebrafish; D049452:Green Fluorescent Proteins