title: Differentiation of embryonic stem cells into insulin-producing cells promoted by Nkx2.2 gene transfer.
abstract: OBJECTIVE
  To investigate the ability of a genetically altered embryonic stem (ES) cell line to generate insulin-producing cells in vitro following transfer of the Nkx2.2 gene.
  
  
  METHODS
  Hamster Nkx2.2 genes were transferred into mouse ES cells. Parental and Nkx2.2-transfected ES cells were initiated toward differentiation in embryoid body (EB) culture for 5 d and the resulting EBs were transferred to an attached culture system. Dithizone (DTZ), a zinc-chelating agent known to selectively stain pancreatic beta cells, was used to detect insulin-producing cells. The outgrowths were incubated in DTZ solution (final concentration, 100 microg/mL) for 15 min before being examined microscopically. Gene expression of the endocrine pancreatic markers was also analyzed by RT-PCR. In addition, insulin production was determined immunohistochemically and its secretion was examined using an ELISA.
  
  
  RESULTS
  DTZ-stained cellular clusters appeared after approximately 14 d in the culture of Nkx2.2-transfected ES cells (Nkx-ES cells), which was as much as 2 wk earlier, than those in the culture of parental ES cells (wt-ES). The frequency of DTZ-positive cells among total cultured cells on day 28 accounted for approximately 1.0% and 0.1% of the Nkx-ES- and wt-ES-derived EB outgrowths, respectively. The DTZ-positive cellular clusters were found to be immunoreactive to insulin, while the gene expressions of pancreatic-duodenal homeobox 1 (PDX1), proinsulin 1 and proinsulin 2 were observed in the cultures that contained DTZ-positive cellular clusters. Insulin secretion was also confirmed by ELISA, whereas glucose-dependent secretion was not demonstrated.
  
  
  CONCLUSIONS
  Nkx2.2-transfected ES cells showed an ability to differentiate into insulin-producing cells.
authors: Division of Developmental Biology, Department of Parasitology, Nara Medical University, Kashihara, Nara 634-8521, Japan.
authors: Akira
authors: A
authors: Shiroi
authors: Shigehiko
authors: S
authors: Ueda
authors: Yukiteru
authors: Y
authors: Ouji
authors: Ko
authors: K
authors: Saito
authors: Kei
authors: K
authors: Moriya
authors: Yuko
authors: Y
authors: Sugie
authors: Hiroshi
authors: H
authors: Fukui
authors: Shigeaki
authors: S
authors: Ishizaka
authors: Masahide
authors: M
authors: Yoshikawa
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D006224:Cricetinae; D018014:Gene Transfer Techniques; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins / Q000235:genetics*; D007328:Insulin / Q000096:biosynthesis*; D007515:Islets of Langerhans / Q000166:cytology* / Q000502:physiology*; D051379:Mice; D008815:Mice, Inbred Strains; D013234:Stem Cells / Q000166:cytology*; D014157:Transcription Factors / Q000235:genetics*; D029961:Zebrafish Proteins
chemical_list: D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins; D007328:Insulin; C000715627:Nkx2-2 protein, mouse; D014157:Transcription Factors; D029961:Zebrafish Proteins; C000715630:nkx2.2b protein, zebrafish