title: An efficient experimental strategy for mouse embryonic stem cell differentiation and separation of a cytokeratin-19-positive population of insulin-producing cells.
abstract: OBJECTIVE
  Embryonic stem cells are a potential source for insulin-producing cells, but existing differentiation protocols are of limited efficiency. Here, the aim has been to develop a new one, which drives development of embryonic stem cells towards insulin-producing cells rather than to neuronal cell types, and to combine this with a strategy for their separation from insulin-negative cells.
  
  
  METHODS
  The cytokeratin-19 (CK19) promoter was used to control the expression of enhanced yellow fluorescence protein in mouse embryonic stem cells during their differentiation towards insulin-producing cells, using a new optimized four-stage protocol. Two cell populations, CK19(+) and CK19(-) cells, were successfully fluorescence sorted and analysed.
  
  
  RESULTS
  The new method reduced neuronal progeny and suppressed differentiation into glucagon- and somatostatin-producing cells. Concomitantly, beta-cell like characteristics of insulin-producing cells were strengthened, as documented by high gene expression of the Glut2 glucose transporter and the transcription factor Pdx1. This novel protocol was combined with a cell-sorting technique. Through the combined procedure, a fraction of glucose-responsive insulin-secreting CK19(+) cells was obtained with 40-fold higher insulin gene expression and 50-fold higher insulin content than CK19(-) cells. CK19(+) cells were immunoreactive for C-peptide and had ultrastructural characteristics of an insulin-secretory cell.
  
  
  CONCLUSIONS
  Differentiated CK19(+) cells reflect an endocrine precursor cell type of ductal origin, potentially suitable for insulin replacement therapy in diabetes.
authors.affiliation: Institute of Clinical Biochemistry, Hannover Medical School, D-30623 Hannover, Germany.
authors.forename: O
authors.initials: O
authors.lastname: Naujok
authors.forename: F
authors.initials: F
authors.lastname: Francini
authors.forename: A
authors.initials: A
authors.lastname: Jörns
authors.forename: S
authors.initials: S
authors.lastname: Lenzen
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D002469:Cell Separation / Q000379:methods; D003470:Culture Media; D017931:DNA Primers; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000648:ultrastructure; D005434:Flow Cytometry; D005934:Glucagon / Q000235:genetics; D007328:Insulin / Q000235:genetics*; D053539:Keratin-19 / Q000096:biosynthesis*; D051379:Mice; D010957:Plasmids; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013004:Somatostatin / Q000235:genetics; D014162:Transfection
chemical_list: D003470:Culture Media; D017931:DNA Primers; D007328:Insulin; D053539:Keratin-19; D013004:Somatostatin; D005934:Glucagon