title: Optimization of three-dimensional imaging on in vitro produced porcine blastocysts and chimeras for stem cell testing: a technology report.
abstract: Differential staining is an immunocytochemical staining that visualizes trophectoderm (TE) and the inner cell mass (ICM) of the blastocysts. It is used to determine the blastocyst quality, but could also be a useful tool to assess the integration site of injected cells into the early embryo. This is relevant for testing of presumed pluripotent stem cells. The gold standard for pluripotent stem cells is to test if the cells are capable of contributing to germline chimeras. Differential staining can be used to evaluate the possibility of chimeric contribution; if the cells are located in the area of the ICM they are likely to contribute to the fetus and if they are located in the area of the TE they are likely to contribute to the fetal membranes. In this article, we optimize on methods for embryo staining and mounting so that the exact location of injected stem cells within preimplantation porcine embryos can be evaluated.
authors.affiliation: 1 Section for Veterinary Reproduction and Obstetrics, Department of Large Animal Sciences, University of Copenhagen , Frederiksberg C, Denmark .
authors.forename: Jan Ole Bertelsen
authors.initials: JO
authors.lastname: Secher
authors.forename: Kristine K
authors.initials: KK
authors.lastname: Freude
authors.forename: Rong
authors.initials: R
authors.lastname: Li
authors.forename: Henrik
authors.initials: H
authors.lastname: Callesen
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology*; D002478:Cells, Cultured; D002678:Chimera / Q000196:embryology*; D053595:Embryonic Stem Cells / Q000166:cytology*; D021621:Imaging, Three-Dimensional / Q000379:methods*; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D039904:Pluripotent Stem Cells / Q000166:cytology*; D013194:Staining and Labeling / Q000379:methods; D013552:Swine