The auxetic nucleus: nuclear mechanotransduction and its role in regulating stem cell differentiation

Embryonic stem cells (ESCs) self-renew in a state of pluripotency, meaning they can give rise to all tissue types; therefore, they are very promising for regenerative medicine. We recently discovered they have a very interesting and surprising property. Just as an ESC begins to leave behind this state of pluripotency - i.e. as it differentiates - their nucleus, the large structure in the cell that houses all the genetic material, becomes 'auxetic'. Auxeticity is a property that refers to the response of a material under mechanical stress. Consider that, under mechanical stress, a tensed rubber band becomes thinner, and when a ball is compressed it becomes fatter: this is what most materials do. However, an auxetic material, in contrast to a rubber band, becomes fatter when stretched, and thinner when compressed. This property is highly unique even compared to other cell nuclei, but we found one other cell type that manifests this same property in its nucleus. That cell type is the oligodendrocyte progenitor cell (OPC), which is similar to ESCs in that they are a self-renewing stem cell. In development, OPCs give rise to oligodendrocytes (the myelinating cell of the central nervous system) and in the adult is responsible for generating new oligodendrocytes following demyelination (a unique and clinically important neural regenerative process called remyelination). Both of these stem cells are keys to regeneration, and we believe our studies will shed new light on how they work.
Auxeticity has two important repercussions for the ESC/OPC nucleus. First, it has implications for structure because auxeticity arises from unique structural characteristics (such as the auxetic honeycomb: see https://www.youtube.com/watch?v=vdkYuLsT7Sc). We will use a combination of biotechnology, biological and physics techniques to understand what nuclear structural properties are responsible for auxeticity; in finding this, we will better understand how nuclear structure changes during differentiation, and how these changes might facilitate differentiation. The second repercussion is that auxeticity yields massive volume fluctuations with mechanical stress. Consider that an auxetic nucleus gets fatter when stretched, and thinner when compressed, and it is clear that, unlike most materials, it changes volume considerably with mechanical stress. This, in turn, will cause a large flux of soluble molecules across the nucleus with mechanical stress. Given that, particularly in tissue, stem cells undergo frequent and significant mechanical stress, we believe this is important for how differentiation is regulated. The reason for this is that there are a number of signaling molecules that are necessary for differentiation that are kept outside the nucleus before ESCs or OPCs differentiate. When the mechanically-stressed nucleus significantly swells (we see volume increases in the nucleus of up to 50% with relatively small forces), that will force some of these molecules into the nucleus where they can find their targets. We propose that in this way, auxeticity causes the nucleus to be like a pump for moving molecules across its membrane. We will use the biotechnology we develop to apply mechanical stress to the cells to observe these volume fluctuations and concurrent movement of molecules across the nuclear membrane. We will also use biological techniques to analyse the targets of these molecules to determine if this auxetic effect is causing functional changes in ESCs/OPCs.
The research will impact biotechnology, regenerative medicine and stem cell biology. It will bring to bear new insight into how stem cells work, and how we can investigate them. Using our connections in stem cells, biophysics, and biotechnology, we will widely circulate our results, generating impact in several academic disciplines. Given its high potential for impact and its highly cross-disciplinary nature, the proposed research is highly suited for the portfolio of the BBSRC.

The stem cell field has largely overlooked how mechanical stress affects lineage specification and self-renewal in stem cells, despite the stem cell niche being a dynamic mechanical environment. During development, stem cells encounter and respond to physical forces. There is little current understanding of how we can exploit these processes for controlling stem cell function in the laboratory. Gaining this understanding is the central objective of the proposed research.
The proposed research is inspired by our observations that embryonic stem cell (ESC) nuclear mechanical properties change considerably during differentiation. We recently observed that transition ESCs - ESCs exiting pluripotency - have nuclei that are auxetic. Auxeticity is a remarkable phenomenon rarely seen in biology; it leads to cross-sectional expansion when stretched and contraction when compressed. Auxeticity has profound implications for volume regulation: physiological stresses cause significant volume changes in the auxetic transition nucleus.
We will use our collective expertise to explore functional significance of nuclear auxeticity by focusing on the importance of mechanical stress. We will develop microfabricated devices to apply mechanical stresses to stem cells, and with these devices and a combination of imaging and biological techniques, demonstrate the importance of auxetic nuclei. We will first investigate nuclear structure to find the mechanisms for nuclear auxeticity. We will use these results to demonstrate how mechanical stress is propagated into the nucleus and affects gene expression. We will investigate how the auxetic volume effect mediates differentiation capacity by regulating interactions between signalling pathways and gene expression. Our research will impact the fields of physical and stem cell biology, and our findings will lead to new methods in regenerative medicine by increasing the palette of tools available for controlling stem cells in the clinic.

The proposed research will generate will impact human health by providing novel and effective approaches for directing stem cell fate as the basis for regenerative medicine. Beyond the academic realm, we have identified the primary beneficiaries of the proposed research.
1. Industry: The research will reveal new ways to analyse stem cells with microfabricated technologies. This can be used for novel biomedical diagnostic approaches. This research could impact commercial technologies in stem cell research and regenerative medicine by providing new insight on controlling and detecting stem cell function. This development will be a key nucleus for future economic growth of the biotech sector in the UK. The research can also benefit the materials industry, in that auxeticity is an extremely useful but rare property with great absorptive capacity (these materials are used for bulletproof vests and sponges). Understanding mechanisms for auxeticity in this application can lead to new understanding of how to develop and make new auxetic materials.
2. General public: Our research will identify the role of stem cells for regenerative medicine and generate a fundamental understanding of the process of differentiation. The anticipated availability of appropriate biotechnology promises to have an immense impact on medical practice. Ultimately the efficacy of health care will be improved and the related treatment costs be reduced. This will enhance the quality of life on a national and international level.
Our envisioned pathway to the eventual impact described above will adopt the following route.
Personal contacts with industrial partners: The investigators have close existing industrial contacts, from imaging and biotechnology (KC and UFK) to pharmaceutical (RF). These industrial connections will be used for commercialisation of intellectual property that may arise from this novel stem cell phenotype and any other results from the proposed research.
Conferences: We will work with the Physics of Living Matter initiative at Cambridge to organise a conference on physical biology of development and stem cells. We are currently organising a similar conference at Chichely Hall for the Royal Society. We will also travel to numerous international conferences and seminars to disseminate our results.
Public engagement: Our public talks, for example at regional meetings of the Institute of Physics, have resulted in tremendous feedback: the public is excited to think about biology from this unique perspective. KC and RF recently gave public lectures for the Cambridge Science Festival, both of which generated significant interest, and KC has given public lectures for the Royal Society on physics and engineering principles in stem cells. RF also speaks widely to lay audiences on stem cells and regenerative medicine. We will amplify these efforts, and expand them to include other avenues of public engagement, as we believe that public engagement in science is essential to the future of science and it also focuses the researcher on issues that are important to the public.
Collaboration. We will devise an appropriate series of collaboration agreements with partners to explore with suitable specialists the right way to deliver these advances into the innovation landscape. We are particularly interested in building further collaborations with Dolomite to enable new biotechnology for stem cell applications, Auxetix for new materials technology, and using RF's pharmaceutical contacts for new stem cell technology. We have extensive collaborations in stem cells, neuroscience, nanotechnology and biotechnology which we can rely on to further extend our network.
Exploitation. It is expected that patentable IP will arise from this project. We are already working with Cambridge Enterprise, the University's IP commercialisation subsidiary, on filing patents for the technology and at the point where proof-of-premise is established.