Anti-KRT18 monoclonal antibody, clone D15

Placental defects are common in bovine embryos produced using assisted reproductive techniques. A proper understanding of the events leading to inner cell mass (ICM) and trophectoderm (TE) specification could help identify the origins of such developmental failures. We focused on caudal-type homeobox transcription factor 2 (CDX2) since it has a specific role during TE differentiation in mouse embryos. Of all the preimplantation stages analyzed, CDX2 protein was present only at the blastocyst stage. To further understand the roles of CDX2 during bovine development, we depleted CDX2 mRNA; despite a significant loss of detectable protein, embryos were able to form blastocysts at the same rate as controls. Embryos lacking CDX2 did not show abnormalities in the number of TE, ICM, or total cells in the blastocyst. Expression of the developmentally important genes SOX2, POU5F1, and NANOG, or TE markers such as IFN-T and KRT18 were not affected by the reduction in CDX2 levels, nor was the localization of SOX2 and POU5F1 protein. Using a functional barrier assay, we observed that the TE epithelial layer of embryos lacking CDX2 had lost its integrity. Our results thus indicate that CDX2 is not required for TE formation during bovine development; nevertheless, it is necessary for maintaining TE integrity. Mol. Reprod. Dev. 81: 962-970, 2014. (c) 2014 Wiley Periodicals, Inc.

Are human trophectoderm (TE) cells committed or still able to develop into inner cell mass (ICM) cells? Human full blastocyst TE cells still have the capacity to develop into ICM cells expressing the pluripotency marker NANOG, thus they are not yet committed. Human Day 5 full blastocyst TE cells express the pluripotency markers POU5F1, SOX2 and SALL4 as well as the TE markers HLA-G and KRT18 but not yet CDX2, therefore their developmental direction may not yet be definite. The potency of human blastocyst TE cells was investigated by determining their in vitro capacity to develop into a blastocyst with ICM cells expressing NANOG; TE cells were isolated either by aspiration under visual control or after labeling with fluorescent 594-wheat germ agglutinin. Further on, aspirated TE cells were also labeled with fluorescent PKH67 and repositioned in the center of the original embryo. Human preimplantation embryos were used for research after obtaining informed consent from IVF patients. The experiments were approved by the Local Ethical Committee and the oBelgian Federal Committee on medical and scientific research on embryos in vitro'. Outer cells were isolated and reaggregated by micromanipulation. Reconstituted embryos were analyzed by immunocytochemistry. Isolated and reaggregated TE cells from full human blastocysts are able to develop into blastocysts with ICM cells expressing the pluripotency marker NANOG. Moreover, the majority of the isolated TE cells which were repositioned in the center of the embryo do not sort back to their original position but integrate within the ICM and start to express NANOG. Owing to legal and ethical restrictions, manipulated human embryos cannot be transferred into the uterus to determine their totipotent capacity. The definitive demonstration that embryos reconstructed with TE cells are a source of pluripotent cells is to obtain human embryonic stem cell olike' line(s), which will allow full characterization of the cells. Our finding has important implications in reproductive medicine and stem cell biology because TE cells have a greater developmental potential than assumed previously. Scientific Research FoundationuFlanders (FWO-Vlaanderen) and Research Council (OZR) of the Vrije Universiteit Brussel. None of the authors declared a conflict of interest.