title: Phenotypic and functional characterization of glucagon-positive cells derived from spontaneous differentiation of D3-mouse embryonic stem cells.
abstract: BACKGROUND
  Glucagon expression is being considered as a definitive endoderm marker in protocols aiming to obtain insulin-secreting cells from embryonic stem cells. However, it should be considered that in vivo glucagon is expressed both in definitive endoderm- and neuroectoderm-derived cells. Therefore, the true nature and function of in vitro spontaneously differentiated glucagon-positive cells remains to be established.
  
  
  METHODS
  D3 and R1 mouse embryonic stem cells as well as α-TC1-9 cells were cultured and glucagon expression was determined by real-time PCR and immunocytochemistry. Functional analyses regarding intracellular calcium oscillations were performed to further characterize glucagon(+) cells.
  
  
  RESULTS
  Specifically, 5% of D3 and R1 cells expressed preproglucagon, with a small percentage of these (<1%) expressing glucagon-like peptide 1. The constitutive expression of protein convertase 5 supports the expression of both peptides. Glucagon(+) cells co-expressed neurofilament middle and some glucagon-like peptide-1(+) cells, glial fibrillary acidic protein, indicating a neuroectodermic origin. However, few glucagon-like peptide-1(+) cells did not show coexpression with glial fibrillary acidic protein, suggesting a non-neuroectodermic origin for these cells. Finally, glucagon(+) cells did not display Ca(2+) oscillations typical of pancreatic α-cells.
  
  
  CONCLUSIONS
  These results indicate the possible nondefinitive endodermal origin of glucagon-positive cells spontaneously differentiated from D3 and R1 cell lines, as well as the presence of cells expressing glucagon-like peptide-1 from two different origins.
authors.affiliation: Institute of Bioengineering, University Miguel Hernandez, Elche, Spain.
authors.forename: Nestor
authors.initials: N
authors.lastname: Vicente-Salar
authors.forename: Alfredo
authors.initials: A
authors.lastname: Santana
authors.forename: Pablo
authors.initials: P
authors.lastname: Juan-Picó
authors.forename: Juan A
authors.initials: JA
authors.lastname: Reig
authors.forename: Enrique
authors.initials: E
authors.lastname: Roche
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D053595:Embryonic Stem Cells / Q000166:cytology*; D005934:Glucagon; D051379:Mice; D010187:Pancreatic Hormones / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D010187:Pancreatic Hormones; D005934:Glucagon