Mechanisms of mechanical regulation of stem cell fate

The emergent field of mechanobiology has established that cell behaviours are influenced by the mechanical properties of the surrounding environment. A key consequence of this influence is that cell fate can be directed by the microenvironment: human mesenchymal stem cells (huMSCs), for example, adopt an adipogenic lineage (i.e. form fat tissue) when cultured on soft materials, but are osteogenic (i.e. form bone tissue) on stiff materials. The well-characterised stiffness sensitivity of huMSCs has advantages for regenerative medicine, where the ability to direct lineage by mechanical stimulation has sparked investigations into potential applications for tissue engineering. The principles of cellular mechanosensing, though, have more general and fundamental biological importance: in embryogenesis and development, tissues must stiffen to enable them to be robust to their functions; in healthy tissues, mechanical homeostasis must be maintained; and in ageing and diseases such as fibrosis, a break-down in mechanical homeostasis can contribute to pathology.

Forces are transmitted from the microenvironment into cells (and vice versa) through integrin adhesion complexes (IACs) embedded in the cell membrane. These complexes bind to proteins in the extracellular matrix (ECM) and interface with the structural proteins of the cytoskeleton. Within the cell, the cytoskeleton is in turn tethered to the nucleus by the LINC (linker of nucleo- and cytoskeleton) complex, which spans the nuclear membrane and is attached to chromatin via the nuclear lamina. There is therefore considered to be a continuous conduit of mechanical linkage between the microenvironment and the cellular centre of transcriptional regulation. The process of converting mechanical stimulation into biochemical signalling is referred to as mechanotransduction. As the field of mechanobiology has become established, a number of key mechanotransduction pathways have been identified; however, these have typically been identified through the activities of single protein entities. Here, we propose a systematic and integrated investigation into protein interactions across the entire mechano-transmission pathway, applied to examine huMSCs as they respond to changes in environmental stiffness. A combination of hypothesis-led and synthetic, global approaches will enable us to address the following objectives:

(a) Using a proximity labelling approach, coupled to mass spectrometry proteomics, we will produce a catalogue of the proteins that interact with each component part of the cellular mechano-transmission pathway (including IACs and the LINC complex).

(b) We will then quantify how protein interactions with component parts of the cellular mechano-transmission pathway are altered in huMSCs cultured on soft, intermediate and stiff materials. This will enable us to build a candidate list of protein interactions involved in mechanosensing.

(c) We will test the importance of the identified protein interactions by introducing mutations to remove them or inhibit their activity. Consequences of impaired mechano-sensitivity will be assessed by determining whether huMSCs are still able to undergo stiffness-directed commitment to adipo- and osteogenic lineages.

The advances that we hope to make with this project will not only serve as a basis for more detailed investigation of the links between the cell microenvironment and gene expression, but will also enable strategies to be developed to control cell behaviour in tissue engineering and therapeutic applications.

The mechanical properties of the cell microenvironment make an essential contribution to cell fate decisions, both during normal differentiation and in age-related diseases. An understanding of the mechanisms of mechanosensing would therefore be a pivotal step in the quest for strategies to modulate cell plasticity, normalise aberrant tissue behaviour and guide tissue engineering.

Many components of the multimolecular structures involved in extracellular matrix (ECM)-driven mechanotransduction have been identified, including integrin adhesion complexes (IAC) at the cell-ECM interface and the linker of nucleo- and cytoskeleton (LINC) complexes at the cytoplasm-nuclear membrane interface. However, our current understanding of the entire signal transduction process is insufficiently integrated.

In work leading up to this proposal, we have defined the first in situ IAC network in fibroblasts using multiplexed proximity biotinylation (BioID). The changes in the network induced by substrates of varying rigidity have also been determined. This has led to a new model for global mechanosensing. We now aim to extend this work to determine how mechanical signals are propagated from the ECM to the nucleus, and to link these mechanisms to cell fate decisions in human mesenchymal stem cells (HuMSCs).

Our objectives will be to:
(a) Define the HuMSC IAC adhesome and LINC complex interactome using multiplexed proximity biotinylation in conjunction with mass spectrometry.
(b) Elucidate the changes in IAC and LINC complex composition that occur in response to varying extracellular rigidity and generate mechanistic hypotheses to explain the changes.
(c) Determine the mechanisms of HuMSC mechanotransduction that link to lineage commitment.

The advances that we hope to make will lead to more detailed investigation of the links between the cell microenvironment and gene expression, and enable translational strategies to be developed for modulation of cell fate in vivo.

The beneficiaries of this study will be:

1. Pharmaceutical and biotech industries. The increasing recognition of the importance of mechanotransduction in control of proliferation and differentiation means that our work will be relevant for companies combatting a range of fibrotic diseases. The improved understanding of the mechanistic links between the cellular microenvironment and cell fate determination that will arise from this project could also inform the design of synthetic matrices for tissue regeneration applications. Should such targets be identified, we will liaise with our institutional technology transfer company and forge links with industrial partners with the aim of developing such technologies. (5-10 years)

2. Organisations and companies recruiting scientifically trained staff, including both public and private sectors. The researcher co-investigator funded by this work will develop their training and expertise, as well as supervising school, undergraduate and postgraduate students. Thus, the work will directly benefit a new generation of scientists. After the completion of the work, the researcher co-investigator will be able to contribute to the scientific economy of the UK by applying the skills gained in the project, whether in public or private sectors. The named individual is beginning to make the transition to independence and the experience he will gain during this project (technical, supervisory and project management) will be tailored to this goal.

3. The general public. Through engagement with the public through talks, websites, social media and general audience publications we seek to communicate the excitement and beauty of scientific research. Our work will generate compelling images that serve as an important starting point for public engagement with biomedicine. We will submit such images to competitions (e.g. Nikon/Wellcome Trust) to reach the widest audience.

Impact deliverables and milestones

A principal milestone will be the publication of research articles in high quality scientific journals and, before this, in bioRxiv. This will enable our findings to be made available to the community as rapidly as possible. We will aim to publish papers as soon as possible, but likely in the second half of the project. Faculty press officers will be informed of any publications so that press releases can be prepared and disseminated. All raw data, particularly from mass spectrometric datasets, will be made available to other researchers at the point of publication. Our findings will also be presented to the field via oral presentations at national and international scientific conferences, with our top choices being the Gordon conferences on Fibronectin, Integrins & Related Molecules and Adhesion Signaling. Our Faculty website will be used as a portal to disseminate findings to researchers, students, commercial companies, and the public at large.