Mechno-regulation of genome function to direct stem cell fate

Lead Research Organisation: Queen Mary, University of London
Department Name: Sch of Engineering and Materials Science

Abstract

A growing number of pathological conditions are associated with inappropriate or defective cellular sensing of their mechanical environment. Adult mesenchymal stem cells (MSCs) provide a promising cell source for many regenerative therapies although relatively little is known concerning the mechanisms through which mechanical stimuli are transduced into regulatory signals within the cell.

Cellular behaviour is largely regulated by gene expression, which is directed from within the nucleus through transcription of DNA. DNA is packaged within the nucleus as chromatin. The compaction state of chromatin impacts gene transcription, and can result in gene silencing. This compaction state is controlled by a number of histone proteins, and is further influenced by the nuclear lamina, a fibrillar matrix within the nucleus lining the inner nuclear membrane. This controls gene silencing in DNA situated near the edge of the nucleus, and is connected via a complex of proteins (the LINC complex) to the cytoskeleton; the cells skeleton which provides the cell with structure. During stem cell differentiation, the nucleus has been shown to remodel, with alterations to nuclear stiffness and the nuclear lamina; potentially influencing gene transcription. Furthermore, if the nucleus stiffness and the connections between the nucleus and cytoskeleton, the LINC complex, are modulated, force transfer to the nucleus may be altered.

This project will address the concept that the nucleus acts as a sensor for mechanical stimuli. It will also address the hypothesis that the role of the nucleus as a mechanosensor changes as a cell progresses along its differentiation route. We will investigate the mechanisms through which mechanical stimuli can induce changes in nuclear organisation and stem cell differentiation. By fully characterising changes in the mechanical properties, protein composition, nuclear architecture and epigenetic signature of biophysically stimulated MSCs as they undergo differentiation, we will identify key pathways responsible for the alteration of cellular mechanosensitivity. These can then be targeted to repair defective mechanosensitivity in diseased or aged cells.

The results of this work will have far reaching implications for our understanding of how cells respond to mechanical stimulation, and will impact strategies for cell based regenerative medicine and musculoskeletal repair.

Technical Summary

Bone marrow derived mesenchymal stem cells (MSCs) provide a promising cell source for a range of regenerative medicine therapies. We have preliminary data indicating that nuclear architecture and cell and nuclear mechanical properties change significantly with differentiation, and are further modulated by biophysical stimulation. We hypothesise that the nucleus as a mechanosensor in the biophysical regulation of stem cell fate.

This project aims to determine the factors behind differentiation-induced alterations to the MSC mechano-phenotype. We will compare human MSCs with and without biophysical stimulation before and after differentiation to elucidate biomechanical, compositional and structural differences in nuclear architecture and the LINC (Linker of cytoskeleton and nucleoskeleton) complex which may modulate mechanosensitivity and cell mechanics.

Analysis of nuclear strain transfer in conjunction with cytoskeletal and nuclear architecture modulation will enhance our understanding of mechanotransduction mechanisms within the cell. Quantitative proteomic analysis will characterise changes in cytoplasmic and nuclear composition with differentiation allowing us to identify potential targets responsible for the modulation of mechanosensitivity. Quantitative data on force transfer across the LINC complex from a nesprin force sensor will provide unique insights into how nuclear mechanoregulation changes with differentiation. Finally, characterisation of biophysically induced epigenetic alterations will identify the mechanisms through which repeated biophysical stimulation can instil an epigenetic mechanical memory within the nucleus.

Planned Impact

Disease and ageing have been associated with abnormal or defective cellular mechanobiology, leading to an inappropriate mechano-response and catabolic signalling. As a result, the focus of this project is not on the development of a therapy, but the understanding of MSC mechanobiology, specifically the mechanisms responsible for alteration of the mechano-response with differentiation. If we are to move the findings of this work toward translation, our primary beneficiaries are other academic researchers, clinicians, biotech and the pharmaceutical industry. We will of course also ensure the general public engage with this exciting stem cell mechanobiology research.

This programme will provide an improved understanding of the mechanisms behind mechanotransduction and how they regulate stem cell differentiation. Given that mechanical forces impact the growth and form of practically every tissue within the human body, defining a role for the nucleus as a mechanosensor will have potential implications across all eukaryotic cell types; for example to better understand and prevent the mechanosensitive initiation and spread of diseases such as prostate cancer. While this work relates primarily to MSCs, it may elucidate more widely applicable cellular signalling mechanisms. Armed with this knowledge, we can begin to explore therapies aimed at restoring the mechanosensitive regenerative potential of diseased and aged MSCs, first for in vitro tissue engineering techniques, and perhaps later for the stem cells residing within our bodies.

We anticipate considerable long term societal benefits to patients suffering from a range of musculoskeletal and orthopaedic ailments. The enhanced understanding of MSC mechanotransduction will lead to improvements in both mechanical conditioning regimes used to engineer replacement tissues, and post-operative rehabilitation regimes. In addition to health, improved treatments bring economic impacts as a result of less post-operative revisions and less time out of work for patients. Furthermore, the parallels drawn between this work on nuclear mechanosensitivity, and debilitating nuclear envelope related diseases may further progress toward the treatment of diseases including Hutchinson Gilford Progeria syndrome and Emery-Dreifuss Muscular Dystrophy.

Towards the end of the project, we will focus on the exploitation of the findings with a focus on R&D investment. In characterising the mechanisms behind alteration of the mechano-response in stem cell differentiation, we will identify pathways, the modulation of which has the potential to rescue diseased or aged cells with abhorrent mechanical signalling. This will be of interest to gene therapy and pharmaceutical companies. There is also potential for collaboration with tissue engineering companies as the findings of this work will enhance the potential of MSCs for these therapies. With applicable skills present among the applicants, we would remain closely involved in the development of any industrial collaboration. In this regard, we also ensure maximum impact for the researcher, with potential opportunities to further develop professionally and remain involved in the project exploitation beyond the grant lifetime.

Publications

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Eraifej J. (2017) Does lamin A/C play a role in neuronal differentiation? in INTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY

 
Description Cellular sensing of the mechanical environment (mechanosensing) is key to natural developmental processes such as stem cell differentiation, while numerous pathological conditions are associated with inappropriate or defective mechanosensing. From the outset, the aim of this work was to explore the biomechanical role of the nucleus and its connection to the cytoskeleton in regulating cell signalling, using stem cell differentiation as a model system in which gross alterations in cell shape and structure associated with distinct lineages have the potential to drastically alter the role of mechanical signals in directing cell behaviour.

Indeed, mesenchymal stem cell differentiation is associated with changes in the shape of the cell's nucleus, which are mediated by changes in overall cell shape and cytoskeletal architecture. These changes in shape are associated with changes in the stiffness of the nucleus which can be attributed to both changes in the organisation and compaction of chromatin (packaged DNA) within the nucleus, and expression and organisation of lamin A/C, an intermediate-filament protein which localises to the nuclear envelope with differentiation. Furthermore, when the membrane on which these cells are cultured is subjected to cyclic strain, epigenetic changes to chromatin are lineage dependent, with significant increases in histone 3, lysine 27 trimethylation (H3K27me3) observed in chondrogenic differentiation.

A particularly interesting finding from this work relates to the lineage dependent strain response in differentiating stem cells. When strain was applied to mesenchymal stem cells undergoing chondrogenic (cartilage specific) differentiation, a distinct organisation and orientation of the nucleus was observed which provides a unique mechanism through which the cell controls strain transfer from the cytoskeleton to the nucleus involving upregulation of the proteins responsible for connecting the cytoskeleton to the nucleus (SUN1 and SUN2). This unique and potentially impactful finding has been further developed through omics approaches employing phosphoproteomics to examine cell signalling pathways, and RNA-sequencing to assess the role of this phenomenon in strain-mediated gene expression. A manuscript describing these results is in preparation. These results have the potential to alter our fundamental understanding of the role played by the largest and stiffest cellular organelle in directing mechanotransduction (Thorpe and Lee, 2020 Nucleus).

In order to explore the impact of cytoskeletal stiffness on cellular mechanobiology, we also conducted a range of experiments in activated cancer-associated fibroblasts. Like stem cells, these cells undergo drastic changes in their shape, structure and role in cancer, and through pharmacological targeting of G-protein coupled oestrogen receptor, we were able to reprogram these cells to reduce cytoskeletal tension (Cortes et al., 2019, Oncogene; Cortes et al., 2019, Hepatology) with the potential to normalise the tumour microenvironment (Cortes et al., 2019a, EMBO Reports; Cortes et al., 2019b, EMBO Reports) and prevent metastatic transformation of pancreatic cancer cells (Rice et al. 2020 Cancers).

However, perhaps the most significant finding of this work relates to a new mechanotransduction pathway. In exploring force balance between the nucleus and extracellular matrix, we focussed on focal adhesions and found that syndecan-4, a cell surface extracellular matrix receptor common to all cell types with expression changes occurring in differentiation and pathology, acts to directly transduce mechanical signals from the extracellular space, leading to a signalling cascade resulting in cell stiffening and downstream changes in gene expression (Chronopoulos, Thorpe et al., 2020, Nature Materials). The elucidation of this cascade has identified mechanisms which can be pharmacologically targeted to alter cellular mechanotransduction with potential uses in a host of disease scenarios.
Exploitation Route The outcomes of this work have impacted our understanding of cellular mechanobiology. The implications of understanding the pathways responsible for strain transfer to the nucleus will be of interest to those working on a range of pathologies termed laminopathies including muscular dystrophy and premature ageing where genetic mutations lead to reduced nuclear stiffness. The elucidation of a new mechanotransduction pathway involving syndecan-4 is likely to open many new research directions in both matrix biology and mechanobiology which have largely focussed on integrins as the primary cell-matrix receptor to date. By describing the signalling pathway through which this pathway acts, we have identified targetable mechanisms which could be pharmacologically exploited to modulate cellular behaviour.

In terms of professional impact, the skills developed and outputs gained over the course of this award has enabled the researcher co-Investigator to secure an academic position at University College Dublin, Ireland, where some the implications of this work will be further explored. The reprogramming of cell mechanics and link to tumour remodelling is likely to have significant clinical implications and these are being explored by both Dr del Río Hernández (collaborator) and Dr Thorpe in collaboration with a group of clinicians in St Vincent's University Hospital in Dublin, Ireland.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Mechano-chemical regulation of cell mechanics and contractility 
Organisation Imperial College London
Department Department of Bioengineering
Country United Kingdom 
Sector Academic/University 
PI Contribution My research team provided intellectual expertise and conducted experiments relating to the mechanoregulation of cell contractility in fibrotic scenarios. My research team was also responsible for training of collaborator's staff in the characterisation of mechanosensitive protein signalling pathways.
Collaborator Contribution The collaborator conducted magnetic tweezer experiments to probe cellular mechanobiology. They also provided intellectual input in dissecting the signalling pathways arising from force on syndecan-4 in addition to those involved in fibrotic mechanosignalling relating to the G-protein coupled estrogen receptor.
Impact Publications: DOI: 10.1002/hep.30193. DOI: 10.1038/s41388-018-0631-3. DOI: 10.15252/embr.201846556. DOI: 10.15252/embr.201846557. DOI: 10.1038/s41563-019-0567-1 DOI: 10.3390/cancers12020289 Multidisciplinary: Collaborator, Dr del Rio Hernandez, provided biophysics expertise. We provided biological and bioengineering expertise.
Start Year 2017
 
Description Bioengineering school visit 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact The Researcher Co-Investigator conducted a visit to girls school in Greenwich, London, to stimulate interest in STEM subjects and biomedical engineering. He introduced the project research via a question and answer session on stem cells, followed by a demonstration of cartilage damage and repair using a bovine hock joint. To finish he played a guess the implant game where students had to guess where in the body a set of implants would go along with their respective function. Each session was 40 minutes with 4 sessions reaching approx. 130 students of approx. 15 years. The teachers then set the students an activity based on the introduction to bioengineering session. Results from this activity quantitatively demonstrated that students had engaged with the subject, knew more about stem cells, and were aware of the concept that physical stimuli to direct cell behaviour.
Year(s) Of Engagement Activity 2017
 
Description Centre of the Cell - Careers in Science & Health Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Researcher Co-Investigator facilitates a STEM learning class each year with students at GCSE level where he talks about his research and career route. This is with a local disadvantaged but academically strong school which consistently supplies students to Queen Mary University. The aim is to increase student engagement with STEM subjects in their A-levels and subsequent plans for further education or employment.

Numbers of students pursuing STEM careers as a result of this interaction are not available, although anecdotal evidence from teachers suggests this is a positive initiative.
Year(s) Of Engagement Activity 2014,2015,2016,2017,2018
 
Description Centre of the Cell Show 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact The Researcher Co-Investigator acts as a scientist STEM ambassador to facilitate engagement with diverse groups of students on a regular basis (every 3-4 months) while visiting the Centre of the Cell centre which is part of Queen Mary University of London. This science education centre is aimed at provoking interest and developing understanding of cell biology and behaviour in children and teenagers aged approx. 7-18 in order to:
>Inspire the next generation of scientists and healthcare professionals
>Stimulate interest, excitement and dialogue about biomedical research
>Raise aspirations, especially in our local community
>Widen participation in further and higher education
>Improve health and wellbeing in our local communities
>Create a local, national and global centre of excellence in Public Engagement

The event involves a short presentation from the Researcher Co-Investigator on his job and career, facilitation of learning through provision of real life examples in the education centre, and fielding of questions relating to science, the project research, and careers in science. Overall the session lasts 90 minutes. School groups interacted with hail from London, across the UK, and Europe.
Year(s) Of Engagement Activity 2012,2013,2014,2015,2016,2017,2018,2019
URL https://www.centreofthecell.org/
 
Description Organ-on-chip workshop - London Science Museum Lates 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Large interactive workshop based around the concept of an 'Organ-on-a-chip shop' as an installation at London Science Museum Lates. This Science Museum Lates night celebrated the launch of the museum's new Wellcome Medicine Galleries and attracted over 4,000 visitors, 300 of which actively participated in our workshop. Museum visitors and researchers chatted about this emerging technology at the shop counter - before being seated in the café to design and make their own organ on a chip keyring. Using a variety of "ingredients" such as coloured beads, pens and tapes - representing the key cell types, 3D environments and mechanical forces present within the organ - visitors created their own lungs, brains, livers and joints, which, when miniaturised, were turned into key rings for them to take home. This was organised through my participation in the Organ-on-a-Chip Network which is part of a major new Research Councils UK (RCUK) venture called Technology Touching Life involving joint research council funding, which aims to foster interdisciplinary research into innovative technology in the health and life sciences. Involved 16 volunteers from across the network.
Year(s) Of Engagement Activity 2020
URL https://www.organonachip.org.uk/news/4629/the-organ-on-a-chip-shop-a-science-museum-highlight
 
Description Pint of Science talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact The Researcher Co-Investigator was invited to give a 20 min public lecture to engage and inform the public on work of the project and wider implications for public health and wellbeing. The talk was held as a ticketed event in a pub in central London with ticket purchase open to all via the Pint of Science festival. The talk sparked significant discussions with members of the public from non-science backgrounds which extended over hours.
Year(s) Of Engagement Activity 2017