The Roadmap of Human Pluripotency

Early embryos of humans and other mammals contain a group of cells called the epiblast. Epiblast cells are pluripotent, meaning that they can generate all the cell types of the developing embryo. How they do this is one of life's great mysteries.
Pluripotent cells are present for up to two weeks in the embryo at the end of which they develop into founder cells for different tissues and organs. In the laboratory we have found ways to keep epiblast cells in a pluripotent state. They continue to multiply and are therefore called pluripotent stem cells (PSCs). PSCs remain similar to epiblast in the embryo and are able to make other types of cells. They provide a unique system for studying development of the early embryo, which cannot be investigated in the womb. The capacity of PSCs to differentiate into all tissue types also offers an unmatched resource for medical research into genetic disease and for drug testing and regenerative medicine. However, to realise the full utility of PSCs requires faithful reproduction of epiblast development in the laboratory.
In our current research we have discovered that the earliest stage of PSC, termed naïve, go through an extended process of maturation before they are competent to become other types of tissue. Competence is the ability to receive and translate instructions to differentiate. Intriguingly, naïve PSCs acquire the competence to make different tissues in a sequence rather than all at once as previously assumed. We hypothesise that successive changes in epiblast competence are key to patterning the human early embryo and may be harnessed for directing PSC lineage choice more efficiently. Therefore, the goal of this research programme is to understand and ultimately to command the flow of lineage competence in human pluripotency.
To reach our goal requires addressing four questions:
1.What is the roadmap to competence?
2.How are signalling inputs integrated to steer competence and induce cellular specification?
3.How are different lineage competences encoded and enacted?
4.Can lineage induction be better optimised and benefit downstream differentiation?
We will use the tools of advanced stem cell culture, including a new model of the early embryo, and apply next generation sequencing technologies, single cell biology techniques and computational methods.
Our first task is to establish the sequence and timing of changes in competence. Associated with this we must expose the underlying patterns of gene expression and determine whether cells follow a single route or a branching path. We must also test for consistency with embryo development. These studies will resolve a central unanswered question in mammalian development: is pluripotency a feature of single cells that are all equally multipotent, or a collective property of a population of cells with different potencies.
For the second question we will profile signalling activity and cell responsiveness during competence progression. We will dissect the central roles of the NODAL-SMAD and FGF-ERK pathways and uncover when and how they drive sequential changes in competence. We will also obtain insight into when and how additional signals, WNT and BMP, are integrated and whether they serve roles in competence or only in induction of differentiation.
How these signals control gene expression to alter cell potency is our third challenge. We will identify key mediators and determine which genes they bind to and regulate. We will characterise interactions with major pluripotency factors, NANOG, SOX2 and OCT4, that switch between genes.
Finally, we will test whether following the route map and logic of the embryo will channel PSC differentiation more reliably for disease modelling.
Together, these studies will illuminate human-specific features of pluripotency and reveal the nature of multilineage competence. This knowledge will inform new approaches to controlling PSC differentiation for fundamental and applied research.

We will deconstruct the cellular and molecular progression of human pluripotency using the experimentally tractable system of pluripotent stem cells in defined culture conditions. We will take advantage of naïve stem cell lines representing the earliest stage of pluripotency, developed and characterised with current MRC funding. As a reference for the human embryo, we will exploit our naïve stem cell blastoid model to generate cultures containing embryonic disc-like structures that capture features of peri- and early post-implantation embryogenesis. We will also use human embryos donated for research.
We will apply our feeder-free culture system to delimit signal input and use flow sorting to purify cells at similar stages in transition. We will generate knock-in fluorescent reporters for live imaging and quantitation of lineage induction by flow cytometry. To measure signalling pathway activities we will use immunoblotting, immunostaining and gene expression assay by qRT-PCR. Additionally, we will deploy fluorescent reporters for live cell detection of signalling dynamics. We will undertake clonal analysis of lineage potential via DNA barcoding. We will generate scRNA-seq time series data to reveal transcriptome landscapes and will integrate lineage analysis using expressed barcodes. Bioinformatics and computational tools will be applied to deconvolve cell trajectories. Histone modification and transcription factor binding profiles will be generated by Cut & Run and integrated with chomatin profiling by ATAC-seq. We will use bioinformatics and experimental approaches to characterise interactions between SMADs downstream of NODAL and BMPs, ETV factors downstream of ERK, and core pluripotency factors. We will use ChIP-PCR for quantitative measurement of binding dynamics at specific regulatory sites. For functional interrogation, genetic perturbations generated via CRISPR/Cas9 will include rapid and reversible protein elimination using degron technology.