Characterisation of bovine epiblast-derived outgrowth colonies
Esben Oestrup A , Jakob Gjoerret B , Kirsten Schauser A , Mette Schmidt C , Vanessa Hall A and Poul Hyttel A DA Department of Basic Animal and Veterinary Sciences, Faculty of Life Sciences, University of Copenhagen, Bülowsvej 17, DK-1870 Frederiksberg C, Denmark.
B Dako Denmark A/S, Produktionsvej 42, 2600 Glostrup, Denmark.
C Department of Large Animal Sciences, Faculty of Life Sciences, University of Copenhagen, Bülowsvej 17, DK-1870 Frederiksberg C, Denmark.
D Corresponding author. Email: poh@life.ku.dk
Reproduction, Fertility and Development 22(4) 625-633 https://doi.org/10.1071/RD08300
Submitted: 9 December 2008 Accepted: 21 October 2009 Published: 11 March 2010
Abstract
The aim of the present study was to characterise bovine epiblast-derived outgrowth colonies (OCs) with respect to the embryonic origin of their cellular components. Epiblasts were isolated mechanically from bovine Day 12 embryos. Epiblasts were cultured on feeder layers of SNL cells (neomycin-resistant leukaemia inhibitory factor (LIF)-producing STO cells) in Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium supplemented with 15% fetal calf serum, 5% KnockOut Serum Replacement, LIF, basic fibroblast growth factor, non-essential amino acids (NEAA) and nucleosides. Samples were fixed on Days 4, 6 and 8 of culture and processed for immunocytochemistry and transmission electron microscopy. Epiblasts formed OCs consisting of a central core of epiblast-like cells with a basal plate of flattened cells extending outwards from the core. The cells of the core showed nuclear octamer-binding transcription factor 4 (OCT4) staining, indicating an epiblast origin, and some also stained positive for cytoplasmic vimentin. Adjacent cells were linked by tight junctions towards the surface of the colony and rested on a basal lamina. The cells of the basal plate predominantly stained for α1-fetoprotein (AFP), indicative of a possible hypoblast origin. Only a few cells scattered within the basal plate exhibited cytokeratin 8 staining, indicating a trophectoderm nature. The intensity of OCT4 and vimentin staining within the core had decreased by Day 8 of culture. In conclusion, OCs derived from bovine Day 12 epiblasts display a central core of OCT4-stained cells of a potential epiblast origin surrounded by a basal plate of mainly AFP-stained cells of a potential hypoblast nature.
Additional keywords: embryonic stem cells, hypoblast, octamer-binding transcription factor 4 (OCT4), pluripotency, trophectoderm.
Acknowledgements
The authors express their sincere thanks to Ms Jytte Nielsen and Ms Hanne Holm for valuable technical assistance. This work was supported by the Danish Agency for Science, Technology and Innovation.
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