Dynamic transcription factor function in control of pluripotent cell sub-states

Stem cells have two defining features; they can divide symmetrically to produce cells functionally identical to themselves and can specialise into the more mature cell types that carry out our bodies' functions, a process called differentiation. To preserve a functional stem cell population, self-renewal and differentiation must be balanced.

The most versatile mammalian stem cell that can grow in the lab has the ability to differentiate into all adult body cells and is called a pluripotent stem cell. Recently, scientists have found that different types of mouse pluripotent stem cells can be grown in the lab; embryonic stem (ES) cells and epiblast stem cells (EpiSCs). In addition, work from our group has identified molecular heterogeneity within ES cell populations that is related to functional differences between the cells. Specifically, undifferentiated ES cells fluctuate between states in which they have high or low concentrations of a particular transcription factor, which we have named Nanog, and that have, respectively, a greater or lesser likelihood of self-renewal. This fluctuating alteration in the propensity of cells to differentiate may be crucial to balancing the opposing stem cell properties in a population. Therefore, understanding how these reversible states are controlled molecularly is likely to impinge on strategies for achieving predictable, uniform control of differentiation and is thus strategically important.

In this research we will examine the function of pluripotency gene regulators at target genes that critically modulate self-renewal efficiency to determine how the pluripotent population is segregated into cells that self-renew efficiently and cells that have a higher likelihood of differentiation.

We have identified a small set of 64 genes that alter transcription in response to Nanog activity and that represent good candidate mediators of Nanog function. We will ask how these 64 genes contribute to functional heterogeneity and how gene regulators control the corresponding genes.

Aim 1: Test whether Nanog-sensitive genes can fully complement Nanog function.
A prominent Nanog-sensitive target, Esrrb can complement several Nanog functions when added back to cells from which Nanog has been removed but cannot fully complement Nanog function. Therefore, we will test the ability of additional candidates to fully compensate for loss of Nanog in combinatorial assays.
Several candidate genes are repressed by Nanog, so we will test the ability of reduction in the level of these candidates to compensate for loss of Nanog.

Aim 2: Determine the biochemical function of pluripotency gene regulators.
We will determine how Nanog fluctuations arise. We will localise gene regulators across the Nanog gene to determine which of these control Nanog and therefore potentially control partitioning between functional subtypes.
We have found that Nanog protein represses the Nanog gene and we will ask how this happens to find out if simple rules govern how Nanog switches different genes on and off.

Aim 3: Compare pluripotency gene regulator function in vivo and in pluripotent human cells.
We will determine whether functional compensations occuring in culture also occur in the mouse embryo.
Interestingly, some candidate regulators can reprogramme EpiSCs to an ES cell state. Human ES cells are more like mouse EpiSCs than mouse ES cells, so we will test the ability of our candidates to influence the growth properties of human ES cells. This could beneficially simplify and reduce the cost of human ES cell culture.

This work will deliver a deeper, more refined understanding of the mechanisms of action of pluripotency gene regulators in cells in culture and in the embryo.

Pluripotent stem cell self-renewal is governed by a pluripotency gene regulatory network (PGRN) centred on the transcription factors (TFs) Oct4, Sox2 and Nanog. Despite considerable advances in identifying additional PGRN components, a detailed distinction between self-renewal and differentiation mechanisms remains elusive. Nanog is a key switch between states. We have identified 64 Nanog-sensitive genes and will ask how these contribute to Nanog function and how they are regulated by Nanog, via three related aims.
Aim 1: Identify Nanog-sensitive genes that fully complement Nanog function. A prominent Nanog-sensitive target, Esrrb, only partially complements Nanog function. Therefore, we will test the ability of additional targets to fully compensate for loss of Nanog in combinatorial assays. We will identify genes showing co-ordinate responsiveness and determine whether specific co-dependencies distinguish pre- and post- implantation PGRN configurations, making use of new bioinformatic approaches.
Aim 2: Determine the chromatin function of pluripotency TFs. Nanog protein represses Nanog, and this may cause Nanog fluctuations. We will use ChIP to determine the context in which Nanog fluctuations arise and which regulators control Nanog. We will compare regulation of Nanog with other negatively/positively regulated targets to determine how Nanog regulates distinct gene cohorts. We will investigate a novel robust pluripotent state defined by reduced Oct4.
Aim 3: Examine pluripotency TF function in vivo, in EpiSCs and in human cells. We will determine whether dependencies seen in culture occur in vivo. We will investigate how TFs control transitions between pluripotent sub-states, during exit from pluripotency and will determine whether a more naïve human pluripotent cell type can be isolated using lessons learnt from our mouse cell studies. Our work will deliver a mechanistic insight into pluripotency TF action in mouse and human cells in culture and in vivo.

The general public will benefit from the results of the proposed work mainly in four ways:
1) Medical research: the proposed research has potential medical implications in two fields of broad interest that may contribute to enhancing the quality of life: regenerative medicine and reproductive medicine.
Regenerative Medicine: The proposed work has the potential to enhance the current understanding of the principles governing the specification and differentiation of pluripotent cell populations and how these processes are balanced. Since the abilities to promote reprogramming to pluripotency of differentiated cells from patients and the ability to robustly and predictably control the differentiation of the resulting pluripotent cells are key steps in most cellular replacement therapies, the direct investigation of the principles and factors governing these processes are of immediate relevance to the field of regenerative medicine.
Reproductive medicine: understanding control of pluripotent cell development has parallels with development of germ cells. The potential contribution of the outlined experiments to the understanding of the molecular control of germ cell specification cannot be immediately translated into advances of clinical relevance. Nonetheless, a fuller understanding of the molecular control of pluripotent cell function will inform and accelerate parallel studies on the mechanisms of germ cell development. This is of pivotal importance in understanding the process of gamete production, and correcting potential defects observed in this lineage.
2) The proposed research has the potential to allow future development of culture additives that can enhance or eliminate propagation of pluripotent cells in culture. The latter is still a hinderance to the application of pluripotent cells, so potential commercial value exists here.
3) The biological research carried out during the proposed project will contribute towards maintaining the high standard of academic excellence currently enjoyed by the MRC Centre for Regenerative Medicine. This will be reflected in the ability of the MRC Centre for Regenerative Medicine and the University of Edinburgh to offer educational opportunities for undergraduate and post-graduate student training.
4) The conceptual advances and tangible material, such as pictures, diagrams and illustrations generated to present the results of the proposed experiments will add to the resources used by our science communication staff during outreach activities aimed at raising awareness of the latest advances in the field of stem cell biology and regenerative medicine amongst diverse audiences within the general public.
Direct academic beneficiaries of the proposed research will include academic researchers interested in the control of pluripotent stem cell populations. More broadly, academic beneficiaries will also include scientists operating in the fields of development, embryonic stem cell biology and reprogramming to pluripotency. Scientist active in the field of embryonic development or interested in the study of gene regulation and epigenetics, as well as members of the medical community will also benefit from the conclusion drawn from the proposed research. The pathway towards academic impact will be based on the classical instruments of scientific communication. Publication of our results in peer reviewed journals and presentations at international scientific meeting of relevance to the field will be the preferred means of communicating the results and conclusions of the proposed project. More specifically, the academic beneficiaries will be the scientists conducting the research, as well as their colleagues and collaborators. Staff funded on the proposed project will develop transferrable professional, analytical and communication skills, as well as specific scientific and technical skills that will contribute to their development and future prospects.