Unravelling the role of beta-catenin in ground state pluripotency

Stem cells are defined by two properties: they can proliferate indefinitely producing cells identical to themselves (self-renewal property), and can specialise (differentiate) into mature cells types (pluripotency property). In adults, stem cells, found for example in the bone marrow, have mainly a repair function in case of injury. Adult stem cells are currently used also in medical therapy; a typical example is bone marrow transplant for leukemia treatment. The major limit of using adult stem cells for medical purposes is the low availability, and the difficulty to expand them in culture maintaining their features intact. Moreover, adult stem cells have a poor regeneration range, as they are unable to differentiate into all desired cell types.

Such issues were overcome thanks to the discovery of embryonic stem cells (ESCs): isolated from blastocysts, ESCs can be cultured in vitro indefinitely, and can give rise, under the appropriate conditions, to every cell type. Also, in 2006 the Nobel Prize winner Yamanaka made an astonishing discovery: somatic cells can be reprogrammed back to a stem-like state, obtaining the so-called induced pluripotent stem cells (iPSCs). ESCs and iPSCs were thought to be a great promise: many believed that their discovery would have fuelled an impressive expansion of regenerative medicine, gene therapy and personalised medicine fields in the short term, ultimately leading to the solution of many health-related problems. So far, only a part of this promise has been fulfilled, mainly because of the scarce knowledge we have about the basic biological processes that make these cells so special.

In this research, we aim at improving our understanding of the molecular processes that orchestrate pluripotency focusing on beta-catenin (b-catenin), a pivotal protein in stemness maintenance. Indeed, we and others reported a central role of b-catenin in somatic cell reprogramming, differentiation, pluripotency and tissue regeneration in vivo. Still, the mechanisms through which b-catenin controls pluripotency of embryonic stem cells are highly debated.

We will unfold this complexity using an interdisciplinary approach. Using cutting-edge technology, we will engineer mouse ESCs (mESCs) in which b-catenin can be modulated in a number of way (amount of protein, temporal dynamics, ability to regulate other genes). Subsequently, we will perform experiments to link specific b-catenin perturbed behaviours to mESCs self-renewal and differentiation ability. Finally, we will use potent computational approaches to understand how b-catenin interacts with other genes important for pluripotency.

The project will provide a vital step of innovation and knowledge to the embryonic stem cells biology field: the results will reveal how b-catenin regulates the balance between pluripotency and differentiation, and indicate how to drive specific cell-fates in vitro.

In the future, the output of the proposed project could be extended to control b-catenin dependent self-renewal and differentiation of human ESCs and iPSCs, shortening the distances between pluripotent stem cells and their applicative targets. Also, the developed techniques could be used in other biological processes in which b-catenin is involved, including development and cancer.

Mouse Embryonic Stem Cells (mESCs) are pluripotent cells, which can be indefinitely expanded in vitro, and pushed into specific differentiated states by proper stimuli. Under standard culture (serum+LIF), mESC population is heterogeneous in pluripotency gene expression and propensity to self-renewal/differentiate. Conversely, serum-free medium (2i+LIF) establishes ground state pluripotency, characterised by homogeneous pluripotency genes levels.
Ground state cultures rely on Chiron, a drug that, through inhibition of the Gsk3 kinase, stabilises beta-catenin (b-catenin), the effector protein of Wnt/b-catenin pathway. Intriguingly, b-catenin is involved not only in self-renewal, but also in differentiation.
The specific role of b-catenin stabilisation in 2i+LIF is still debated.
We recent proved heterogeneity and oscillations of b-catenin in ground state cultures, despite homogeneity of pluripotent markers. These results motivate the research ideas of this proposal, aimed at assessing the role of b-catenin in ground state pluripotency.

Current ground state cultures with Chiron do not allow:
1) Distinguishing b-catenin dependent from independent effects;
2) Decoupling b-catenin levels from dynamics, due to intrinsic feedback loops;
3) Distinguishing transcriptional and post-translational functions of b-catenin.

Therefore, we will implement in mESCs deleted for endogenous b-catenin an inducible tool, enabling to generate fine-tuned perturbations of exogenous, Chiron-insensitive b-catenin. Differently from previous studies, we will generate a gradient of levels, dynamics and transcriptional activity of exogenous b-catenin, aided by mathematical modelling. We will assess the pluripotency of mESCs perturbed for b-catenin; also, we will determine how b-catenin interacts with the ground state transcriptional network, using a data-driven computational approach.
Outputs of this project will indicate how to perturb mESCs decision-making by b-catenin modulation.

Who will benefit from this research?

In addition to contributing to the advance of the research fields close to the proposal themes, our research has the potential to have an impact on a wider group of other parties, such as:
- Medical research community
- Biomedical industry
- General public
- Research personnel involved in the project
- PI institute

How will they benefit from this research?

Medical research community
The proposed research has potential to have implications in regenerative medicine. Although our research is concerned with ground state pluripotency in mouse, ultimately project techniques and extensions could impact protocols for maintenance and robust differentiation of pluripotent human stem cells. Such advances, necessary for safe application of pluripotent cells, would in the long run have an impact on health and well-being. The pathway to reach the medical research community will be based on the project partners' expertise in regenerative medicine, as well on existing and novel links to researches in UK ad over sea.

Biomedical industry
The demand for stem cells-related products is rapidly growing. The work proposed here has the potential, on the long-term, to provide basis for refined ground state cultures and differentiation protocols, which could be appealing for companies who provide stem cells related products. If during research we will identify exploitation opportunities, we will critically consider the possibility of patentability, with the support of the Bristol University Researcher and Enterprise Development programme (RED).

General public
Stem cell research is a topic of great interest and debate for general public, given potential applications in regenerative medicine, and ethical concerns. We believe that sharing research ideas and results with general public is an honest and clear way is a fundamental part of the scientist work. Therefore, we will disseminate findings and, more generally, research ideas behind the research field of stem cells by taking part to public engagement activities organised by the University of Bristol and the institutes of the project partners.

Research personnel involved in the project
The experimental PDRA (funded by this grant) and the theoretical PhD student (already in the PI's research group) involved in the project will gain a number of cutting edge skills that will help them in their future academic or industrial careers. The PRDA and PhD student are expected to actively interact, and engage with a multidisciplinary research group. A benefit of this proposal is that the PI is experienced in working in both experimental and computational communities, and currently mentors interdisciplinary researchers. Contributing to the training of the next generation scientists, with transferable expertise in more than one discipline, will have a strong impact on UK competitiveness in science and economy. The Regenerative Medicine Laboratories and the Engineering Mathematics Department and the in Bristol, together with the Centre for Genomic Regulation in Spain, will be an excellent environment for all project participants to exchange knowledge, and connect with a network of interdisciplinary investigators.

PI institute
The Engineering Mathematics Department, in which the PI is based, is composed by scientists with a diverse background, and the common aim of tackling complexity in different fields (biology, ecology, robotic, engineering) by combined experimental and computational approaches. The Department has an excellent track record both in terms of publications, and grants awards. The research carried out in this proposal would contribute towards maintaining high standards of academic excellence of the Department, and establishing new interdepartmental connections. Academic excellence will be echoed by the ability of the University to attract talented PhD students and PDRA researches.