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-α-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-α-fetoprotein-specific antibodies to a peripheral cellular layer, suggestive of yolk sac visceral endoderm (VE) formation. Conclusion: 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.

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