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

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.

We recently developed a protocol for derivation of multiple clones of naïve pluripotent human embryonic stem (HNES) cells from a single embryo using serum-free culture medium supplemented with LIF and inhibitors of FGF signalling, GSK3, ROCK and aPKC. We propose to utilise this technology to sample the methylome and gene expression profile simultaneously from single cells of individual founder embryos before, during and after generation of HNES cell lines using the recently published scM&T-seq technology. The data generated will be used to explore the variation of gene expression and associated epigenetic modification, particularly of imprinted genes, between cells at each stage and during the process. In additional experiments, we will expand individual clones arising from cells separated from the same embryo to exploit the possibility to obtain a proportion of clones carrying chromosomal defects of potential medical interest that frequently occur intermixed with normal cells during assisted conception. HNES cells will be used as a model of the implanting human embryo to study the biomechanical changes associated with the transition from apolar inner cell mass to polarised epithelial epiblast. We will examine the expression and distribution of key cytoskeletal proteins during the transition from naïve to primed pluripotency and differentiation, primarily using immunocytochemistry and confocal analysis. For example, we will utilise specific small molecules affecting the actin cytoskeleton, such as Cytochalasin or Jasplokinolide. The readout from these experiments will dictate candidates that will be knocked down to confirm their specific activity and utilised to optimise future culture regimes. Use of compliant hydrogels to which specific extracellular matrix proteins can be tethered will enhance naïve pluripotency and reduce variables in the regime currently imposed by the fibroblast feeder cells currently required.

This project will benefit researchers and practitioners in multiple fields, including stem cell and developmental biology, epigenetics, chromosomal aberrations and assisted conception. The possibility to generate separable clones during the derivation of human pluripotent stem cell lines from a single embryo opens up many new opportunities. It provides a means to investigate in detail the transcriptional changes that may occur during the capture and expansion of self-renewing embryonic cells without having to account for inter-embryo variability. Combining this with the timely development of a dual method to profile the transcriptome and methylome from a single cell will provide a rich resource available to everyone interested in the molecular control of human pluripotency. The important role played by epigenetics in this process, particularly in the context of imprinting, will be of wide interest to stem cell and developmental biologists. Our second objective, to generate multiple clonal stem cell lines from single embryos, has the potential to provide both normal and chromosomally aberrant cell lines on a single genetic background. Such a resource will benefit researchers and clinicians interested in the consequences of congenic syndromes such as Down's, since they will be able to compare directly the behaviour of tissues of interest, which can be generated in culture by directed differentiation protocols, of affected cells versus controls. The lines generated in this project will be made available via the UK Stem Cell Bank. Our third objective is to enhance understanding of the biomechanical processes associated with the early events of differentiation. By investigating the cytoskeletal changes associated with the transition from naïve, dome shaped colonies to primed, epithelial sheets following various chemical and genetic manipulations will be of interest to stem cell and developmental biologists, biophysicists, assisted conception practitioners and patients suffering from repeated pregnancy failure.