Human trophectoderm cells are not yet committed.

STUDY QUESTION: Are human trophectoderm (TE) cells committed or still able to develop into inner cell mass (ICM) cells? SUMMARY ANSWER: 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. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: 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. PARTICIPANTS/MATERIALS, SETTING, METHODS: 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 'Belgian 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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. LIMITATIONS, REASONS FOR CAUTION: 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 'like' line(s), which will allow full characterization of the cells. WIDER IMPLICATIONS OF THE FINDINGS: Our finding has important implications in reproductive medicine and stem cell biology because TE cells have a greater developmental potential than assumed previously. STUDY FUNDING/COMPETING INTEREST(S): Scientific Research Foundation-Flanders (FWO-Vlaanderen) and Research Council (OZR) of the Vrije Universiteit Brussel. None of the authors declared a conflict of interest.

HLA-G expression in human embryonic stem cells and preimplantation embryos.

Human leukocyte Ag-G, a tolerogenic molecule that acts on cells of both innate and adaptive immunity, plays an important role in tumor progression, transplantation, placentation, as well as the protection of the allogeneic fetus from the maternal immune system. We investigated HLA-G mRNA and protein expression in human embryonic stem cells (hESC) derived from the inner cell mass (ICM) of blastocysts. hESC self-renew indefinitely in culture while maintaining pluripotency, providing an unlimited source of cells for therapy. HLA-G mRNA was present in early and late passage hESC, as assessed by real time RT-PCR. Protein expression was demonstrated by flow cytometry, immunocytochemistry, and ELISA on an hESC extract. Binding of HLA-G with its ILT2 receptor demonstrated the functional active status. To verify this finding in a physiologically relevant setting, HLA-G protein expression was investigated during preimplantation development. We demonstrated HLA-G protein expression in oocytes, cleavage stage embryos, and blastocysts, where we find it in trophectoderms but also in ICM cells. During blastocyst development, a downregulation of HLA-G in the ICM cells was present. This data might be important for cell therapy and transplantation because undifferentiated hESC can contaminate the transplant of differentiated stem cells and develop into malignant cancer cells.

Can soluble human leucocyte antigen-G predict successful pregnancy in assisted reproductive technology?

PURPOSE OF REVIEW: To review the predictive value of soluble human leucocyte antigen-G (HLA-G) in embryo culture fluid for implantation, considering origin, structure, function and detection method of soluble HLA-G. RECENT FINDINGS: Soluble HLA-G in embryo culture supernatant has been proposed as a noninvasive marker for the selection of embryos with implantation potential. Controversially, some centres detect soluble HLA-G in none of the culture supernatants of embryos that evolve towards pregnancy, whereas others report it to be mandatory. According to a recently published meta-analysis, the pooled diagnostic accuracy for predicting clinical pregnancy is low. Factors influencing soluble HLA-G results are numerous, for example, embryo culture (single or multiple, washing/denudation steps, medium, day 2 to 5 sample collection), sample preservation to prevent degradation and HLA-G immunoassays (standard, detection limit, reproducibility). Published studies are very heterogeneous. Moreover, the origin of soluble HLA-G in embryo culture fluid is unresolved. From the embryonic genome activation at day 3 onwards, the embryo should be able to produce HLA-G. Before, it is maternally derived, either from the oocyte, follicular fluid or follicular cells contaminating embryo culture. SUMMARY: At present, no studies have been published on the relationship between pregnancy and soluble HLA-G in supernatants from individually cultured and individually transferred embryos using standardized embryo culture and soluble HLA-G immunoassay, sensitive at the picogram level. As such, it remains undetermined whether the pregnancy is induced by an HLA-G-producing embryo. Therefore, the predictive value of soluble HLA-G in embryo culture supernatant for selection of embryos with good implantation potential remains unknown.

Critical role of N-cadherin in myofibroblast invasion and migration in vitro stimulated by colon-cancer-cell-derived TGF-beta or wounding.

Invasion of stromal host cells, such as myofibroblasts, into the epithelial cancer compartment may precede epithelial cancer invasion into the stroma. We investigated how colon cancer-derived myofibroblasts invade extracellular matrices in vitro in the presence of colon cancer cells. Myofibroblast spheroids invade collagen type I in a stellate pattern to form a dendritic network of extensions upon co-culture with HCT-8/E11 colon cancer cells. Single myofibroblasts also invade Matrigel trade mark when stimulated by HCT-8/E11 colon cancer cells. The confrontation of cancer cells with extracellular matrices and myofibroblasts, showed that cancer-cell-derived transforming growth factor-beta (TGF-beta) is required and sufficient for invasion of myofibroblasts. In myofibroblasts, N-cadherin expressed at the tips of filopodia is upregulated by TGF-beta. Functional N-cadherin activity is implicated in TGF-beta stimulated invasion as evidenced by the neutralizing anti-N-cadherin monoclonal antibody (GC-4 mAb), and specific N-cadherin knock-down by short interference RNA (siRNA). TGF-beta1 stimulates Jun N-terminal kinase (also known as stress-activated protein kinase) (JNK) activity in myofibroblasts. Pharmacological inhibition of JNK alleviates TGF-beta stimulated invasion, N-cadherin expression and wound healing migration. Neutralization of N-cadherin activity by the GC-4 or by a 10-mer N-cadherin peptide or by siRNA reduces directional migration, filopodia formation, polarization and Golgi-complex reorientation during wound healing. Taken together, our study identifies a new mechanism in which cancer cells contribute to the coordination of invasion of stromal myofibroblasts.