Defining the prerequisites of naive pluripotent human embryo cells for self-renewal in culture

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Until recently human embryonic stem cell lines were derived following explantation of intact early human embryonic structures into culture. We have recently applied a novel protocol in which the embryonic cells are first separated from one another before culture is begun. By this means, we have generated normal stable stem cell lines that we named 'human naïve epiblast stem (HNES) cells'. There are several advantages with this new approach, since it enables production of individual clones from a single individual. From a developmental biology point of view, separation of the embryonic cells prior to culture prevents the inter-cell communication that drives cell specification, thus retaining them in a 'naïve' state. Also, multiple clones can be produced from each embryo. We propose to utilise this novel opportunity to sample individual clones at various stages during the derivation process, whilst expanding the remaining clones into HNES cell lines. The sampled clones will be separated into single cells, each of which will be profiled simultaneously for gene expression and epigenetic modifiers. This will allow us to track the process of derivation at a molecular level to determine what changes, if any, occur during the transition from embryonic cell to stable HNES cell line. This approach removes the genetic variability between individuals hampering all related studies performed so far. Another objective of this project is to take advantage of the frequent tendency (around 60%) of in vitro fertilised human embryos to exhibit mosaic aneuploidy. For these experiments, we propose to expand each of the colonies from a single explanted embryo separately and check the karyotype of each cell line. The resulting characterised HNES cell lines will then be lodged in the UK Stem Cell Bank for distribution to interested researchers. Most frequent amongst the reported aneuploidies is trisomy of the small chromosomes 21 or 22. Having separate cell lines with and without the chromosomal defect provides an ideal system with which to compare the consequences of aneuploidy in various in vitro-derived tissues as a model for such congenic disorders as Down's syndrome, in direct comparison to unaffected tissues on the same genetic background. We have already shown that our derivation protocol produces embryonic stem cell lines that bear the closest resemblance to cells within the early embryo compared with all other reported lines. Our cell lines are therefore ideal models with which to investigate the early developmental process occurring during and after implantation in the uterus that are inaccessible in vivo. We are particularly interested in identifying the biomechanical changes required to orchestrate the physical processes involved in generation of the early foetus during the period of pregnancy during which the embryo is most at risk of miscarriage. We will investigate ways of altering the structure of the cells to monitor the effects on development in culture. We will use high resolution microscopy to see how important proteins are localised within each cell. These studies will enhance our understanding of human development and provide useful information that may be incorporated in the design of new protocols to produce specific tissues in culture for biomedical research and disease modelling.