title: Human embryonic stem cells form embryoid bodies containing visceral endoderm-like derivatives.
abstract: OBJECTIVE
  The objective of this study was to determine the potential of human embryonic stem (hES) cells to provide an in vitro model of human extraembryonic endoderm development.
  
  
  METHODS
  The hES cell line HES-2 was propagated in Dulbecco's modification of Eagle's medium (DMEM) in the presence of 20% fetal calf serum (FCS) on a mouse embryonic fibroblast feeder layer. Clumps of approximately 50-100 cells were transferred to fresh DMEM and FCS and grown as embryoid bodies (EBs) in suspension culture. EBs were subjected to immunohistochemistry for endodermal, ectodermal and mesodermal specific markers and immunoreactivity analysed by confocal microscopy and on cryosections.
  
  
  RESULTS
  HES-2 cells reproducibly formed spherical EBs after 2-3 days in suspension culture as clumps. EBs continued to expand and by 7 days had commenced cavitation in a highly reproducible and organised fashion. Culture periods longer than 10 days led to cystic structures displaying inconsistent morphological organisation. Immunolocalisation of anti-alpha-fetoprotein-, anti-neurofilament- and anti-CD31-specific antibodies at 7 days of culture provided evidence of regional differentiation of endodermal, ectodermal and mesodermal derivatives in cavitating EBs. Further, of 10 cavitating EBs analysed at 7 days of culture, all displayed immunolocalisation of anti-pan-keratin-, anti-CK8- and anti-alpha-fetoprotein-specific antibodies to a peripheral cellular layer, suggestive of yolk sac visceral endoderm (VE) formation.
  
  
  CONCLUSIONS
  Cavitation, the presence of regionalised cell lineage-specific immunoreactivity and the development of a VE-like peripheral cell layer demonstrate that the HES-2 hES cell line can be induced to undergo EB formation and provide scope to study early human primitive endoderm and yolk sac VE development.
authors.affiliation: Monash University, Monash Institute of Reproduction and Development, Centre for Early Human Development, Clayton, Australia.
authors.forename: Brock J
authors.initials: BJ
authors.lastname: Conley
authors.forename: Alan O
authors.initials: AO
authors.lastname: Trounson
authors.forename: Richard
authors.initials: R
authors.lastname: Mollard
mesh_terms: D000818:Animals; D002449:Cell Aggregation; D002454:Cell Differentiation; D002460:Cell Line; D002871:Chromosome Banding; D004622:Embryo, Mammalian / Q000166:cytology*; D004707:Endoderm / Q000166:cytology*; D005855:Germ Layers / Q000166:cytology; D006801:Humans; D007621:Karyotyping; D051379:Mice; D013234:Stem Cells / Q000166:cytology / Q000276:immunology / Q000502:physiology*; D013997:Time Factors; D014781:Viscera / Q000196:embryology*