Human Pluripotency

Pluripotency is the flexible capacity of individual cells to give rise to all somatic lineages. In the human embryo the period of pluripotency extends for more than 10 days, from emergence in the late blastocyst through to lineage commitment during gastrulation. In mice two types of pluripotent stem cell line derived in vitro are thought to represent respectively the initial naïve condition and a late phase primed for differentiation. Human pluripotent stem cells exhibit attributes of priming, reflected in molecular features such as DNA methylation and, most significantly for application purposes, in variable differentiation biases. Recently, however, we have established human stem cell cultures that exhibit properties anticipated for naïve pluripotency. Here we will further validate naïve identity and enhance the stability and consistency of these cultures. We will then characterise their transition towards lineage commitment. Understanding this process and faithfully recapitulating in utero pluripotency progression may substantially improve the robustness and fidelity of directed differentiation for biomedical applications. In vivo reference material is lacking, however, because the peri- and early post-implantation period is essentially inaccessible in human embryo development. Therefore, exploitation of naïve human pluripotent stem cells offers both a unique challenge as well as a substantial opportunity. The phase of naïve pluripotency in human is only partially conserved with rodents and key factors that support the robust self-renewal of mouse embryonic stem cells are missing. Our first task therefore is to implement both candidate gene studies and genome-wide screens to identify pivotal regulators that can enhance the stability of human naïve pluripotent cells in vitro. We will then optimise the generation of naïve stem cells both by derivation from donated embryos and by reprogramming. In parallel we will establish the conditions for naïve cells to transition to an intermediate, or formative, population that recapitulates the embryonic disk, and subsequently to progress efficiently to lineage specification. We will characterise these conversions by deep transcriptome and epigenome profiling taking advantage on next generation sequencing technology. Having defined the path to lineage specification, we will be positioned to determine the extent to which differentiation biases commonly observed among conventional human pluripotent stem cells may be muted by resetting to naïve pluripotency. Observed restoration of developmental potential will be characterised further to reveal whether "correction" is mediated by transcriptional rewiring and/or epigenome erasure. Our laboratory possesses the experience and expertise for all the stem cell culture and manipulation aspects of this project. We also have outstanding collaborators in domains of early embryology, CRISPR screening, sequencing informatics, DNA methylome, transposable elements, media formulation, physical biology, computational modelling and genome integrity.