Engineering Circadian Biology into Human Induced Pluripotent Stem Cell Organ-on-a-Chip models

British Heart Foundation – 4 year Doctoral Training Programme

Led by Professors Amrita Ahluwalia and Tim Warner and involving 23 named researchers, the BHF DTP Programme provides cohort training leading to a PhD in cardiovascular research.

Super-Resolution Microscopy of live cells in 3D

This project funds the acquisition of a novel super resolution microscope, OMX-FLEX, which will be used to develop a range of new techniques enabled by fast 3D Super-Resolution imaging of live-cells.

Incorporating the circadian clock into Organ-on-a-chip (OOAC) devices

Studies to support the development of more physiologically relevant in vitro model systems which incorporate the circadian clock

University Enterprise Zone

Development of space and infrastructure to expand activities for the incubation of life sciences spin-out and start-up companies.

Does the biological clock within cartilage align to diurnal patterns in activity?

Mechno-regulation of genome function to direct stem cell rate

Mechno-regulation of genome function to direct stem cell rate

Hydrothermal Biomass Upgrade into Carbon Materials and Leuvinic Acid for Sustainable Catalysis - HydroCat Marie Curie (CIG)

SuprHApolymers - Engineering macromolecular self-assembly of hyaluronan (HA)-based glycopolymers with peptides

“SuprHApolymers” project aims to design and synthesize glycopolymers mimicking the composition and structure of hyaluronan (HA), a linear polysaccharide composed of repeating disaccharide units of N-acetyl-glucosamine and glucuronic acid but with many important biological functions. Linear glycopolymers, made solely of GlcNAc or GlcUA sugars (homopolymers) or containing both sugars (copolymers) will be synthesized to study their interaction with synthetic peptides bearing HA-binding motifs (peptide library). The synthesis of HA-based glycopopymers with branched architecture will be also attempted to explore different polymer configurations and to create optimal interactions with peptides. The self-assembly of HA glycopolymers with peptide amphiphiles containing selected HA-binding sequences will be investigated to form de novo peptide-polymer hybrid supramolecular materials with different molecular and macroscopic properties. Finally, the formed functional assemblies (nanostructures and supramolecular gels or films) will be explored for applications in synthetic biology and biomedicine.

Effect of cell age on cell migration and cytoskeletal reorganization’

Effect of cell age on cell migration and cytoskeletal reorganization

CSKFingerprints

Use cytoskeletal morphometrics to characterize cell function, behaviour and pathologies

Novel dynamic self-assembling system - BIOMORPH

The project aims to invetigate the molecular mechanisms between peptides and proteins t create dynamic materials

Optimal Cartilage Regeneration

Augmenting sirtuin activity to drive cartilage regeneration and treat osteoarthritis.

Osteoarthritis (OA) is a highly prevalent disease involving degeneration of articular cartilage and chronic inflammation of the joints, with Worldwide estimates that 9.6% of men and 18.0% of women aged >60 years have symptomatic OA. Prevalence increases with age, such that OA is expected to be the fourth leading cause of disability by the year 2020 (WHO report, 2003). Thus factors that slow the onset or progression of this disease will significantly benefit the well-being of older people.

Multiscale Mechanobiology for Tissue Engineering

Platform Grant Strategic Research Areas Mechanics and mechano-signalling at the sub-cellular, cellular and tissue levels. The response of living cells and tissues to mechanical forces is critical to tissue health and homeostasis. Consequently this field of mechanobiology has enormous potential to be exploited in the development of Tissue Engineering strategies for the regeneration of diseased or damaged tissue. Fundamental to the success of these strategies, is the ability to predicate the effect of physiological mechanical loading on the cellular response. Consequently, a key strategic aim is to develop and use methods to quantify biomechanical and biophysical parameters at a variety of hierarchical levels, within tissues and tissue engineered constructs. Furthermore, it is also necessary to identify the mechanosensory behaviour, such as cytoskeletal dynamics protein trafficking etc, and the important mechanoreceptors and signalling pathways which mediate the biological metabolic and genomic responses to mechanical loading.   Micorenvironmental sensing/manipulation and predictive in silico modelling in 3-D The culture of cells in three-dimensional scaffolds induces the formation of spatial gradients of key nutrients and metabolites, potentially leading to spatial heterogeneity of cell response. The processes underlying this phenomenon are poorly understood and complex, involving tightly regulated interplay between nutrient/transport provision, associated with diffusion/perfusion, and metabolic utilisation or sequestration of soluble bio-regulators by cells. The application of mechanical loading influences these processes, thereby affecting cellular function and neo-tissue elaboration, via direct mechanosensing by cells and also through altered nutrient/metabolite transport. This represents a key strategic research area for the platform grant with the aim of developing novel, non-destructive analytic techniques to interrogate the micro-environmental milieu within 3-D constructs with mechanical perturbation. Mathematical modelling can provide an enhanced understanding of the complex interplay between an array of factors that control neo-tissue growth. Moreover, modelling may be used for control systems in bioreactors. A further key strategic aim is to interrogate the effects of manipulation of the micro-environmental milieu through external manipulation of oxygen levels in combination with mechanical perturbation. This permits the provision of a more physiological mechano/metabolic environmental conditions that will be a powerful tool for tissue engineering, but also for studying patho-physiological processes and by providing more relevant 3-D model systems for drug discovery applications.   Construct technology and advanced tissue processing The translation of basic science to therapeutic application requires the development of novel, efficient and efficacious construct and process technologies, with integrated conditioning and monitoring, to ensure reliable neo-tissue production prior to implantation. Tissue growth, repair and maintenance is defined and sustained through the appropriate distribution of specific biological and biophysical cues, which form microenvironmental domains within the extracellular milieu. This complex interplay between molecular and biophysical signals and the associated cellular response is necessary for functional tissue development. The platform grant may be used to push forward the development of novel scaffold technologies and bioreactor systems, designed to provide defined biochemical and mechanical cues to seeded cells in distinct spatial and temporal domains, thereby creating a defined bioengineered ‘cell-niche’ that controls cell differentiation and activity. One basic strategy might involve heirarchical functionalisation, at the nano- and meso-scales, of natural and synthetic polymeric biomaterials, selected due to their track record for use as components of bioresorbable implants. Differential properties may be achieved at the mesoscale using a variety of complementary synthetic approaches. Cutting edge technologies such as emulsion templating may be utilised to develop porous surface modified biomimetic supports. Ultimately ‘on-demand’ release systems are envisaged, activated by metabolic mediators associated with cell stress and nutrient deprivation. Thorough characterisation of neo-tissue elaboration in the resultant tissue engineered constructs will be achieved using bioreactor systems designed to provide defined biomechanical perturbation and modification of the extracellular milieu via medium perfusion and/or control of the oxygen environment. Platform Grant Flexibility The platform grant provides exciting flexibility enabling new research avenues to be explored within the broad remit of the proposal outlined above. Consequently individuals interested in applying for a post doctoral research assistant (PDRA) position on this platform grant will be encouraged to explore and develop their own research niche. Thus the PDRA positions within the Cell and Tissue Engineering group should provide an extremely attractive spring board to scientific independence and career progression.

Does Warburg energy metabolism contribute to the phenotypic stability of monolayer expanded chondrocytes?

Elucidating the contribution of bioenergetic phenotype to the preservation of the differentiated chondrocyte phenotype in vitro.

The modulation of metabolic phenotype in chondrocytes during monolayer expansion in relation to synthetic phenotypic stability, oxidative stress and proliferative senescence.

Elucidating the role of the chondrocyte bioenergetic phenotype in the preservation of a differentiated synthetic phenotype and senescence in vitro.

Mechanoregulation of nuclear architecture and genome function: A novel mechanism in stem cell fate (Human Frontier Science Program funded)

Gene expression can be regulated through alterations in nuclear architecture, providing control of genome function. Mechanical loading induces both nuclear deformation and alteration in gene expression in a variety of cell types. One putative transduction mechanism for this phenomenon involves alterations to nuclear architecture, resulting from the mechanical perturbation to the cell, to induce altered gene expression. Moreover, remodeling of the nucleus occurs during stem cell differentiation, causing alterations in nuclear stiffness and potentially influencing nuclear architecture-mediated mechano-regulation of transcription. Indeed, a growing body of literature suggests that stem cells respond to mechanical loading differently depending on their differentiation state. Accordingly this project will focus on this aspect of mechanoregulation of genome function, through the following scientific aims and objectives: Scientific aims of the project: To test the following hypotheses 1. Mechanically-induced nuclear deformation causes alterations to the nuclear architecture and chromosomal territories sufficient to affect gene transcription 2. Stem cell differentiation modulates the process set out in hypothesis 1, via differentiation-induced nuclear remodeling that affects the mechanical properties of the nucleus. Specific Objectives: 1. To characterize nuclear deformation induced by the application of static or dynamic mechanical perturbation and to determine the associated rearrangement of chromosomal territories. 2. To assess the mechanical properties of the nucleus in stem cells in their undifferentiated state and during differentiation 3. To assess the mechanism of transfer of strain to the nucleus through the extracellular matrix and cytoskeleton. 4. To conduct genome-wide analysis of mechanosensitive genes and map the relevant loci to chromosomal territories within the nucleus 5. To assess the functional relevance of mechanical-perturbation-induced nuclear deformation on regulation of transcription using 3-D FISH analysis for genes selected from the results of objective 4. 6. To develop an integrated model of mechanosensitive genome regulation that incorporates the mechanical, morphological and transcriptomic data from previous objectives. In order to fulfill the stated objectives the studies will utilize model systems involving human mesenchymal stem cells and induced pluripotent stem cells in conjunction with state-of-the-art techniques for the mechanical perturbation of cells, functional analysis of nuclear organization and biomechanical analysis. Ultimately a mechanistic model for functional mechanoregulation of nuclear architecture will be developed to incorporate elements of the modeling of the mechanical properties of the nucleus in conjunction with functional genomic analysis. The proposed project clearly meets the aims of the HFSP as it focuses on elucidating fundamental and complex biological mechanisms associated with genome regulation and stem cell fate. The focus is on basic understanding, but the outcomes will clearly have relevance to more application driven fields, such as regenerative medicine. The proposal further meets the aims of the HFSP by bringing together scientists from different fields and in doing so will develop new approaches to understanding complex biological systems.

FlowMat Marie Curie (CIG)

The role of membrane-actin adhesion in regulating stem cell viscoelastic properties and blebability during differentiation

This PhD examines how chondrogenic differentiation of human mesenchymal stem cells (hMSCs) regulates the interaction between the cell membrane and the actin cortex, thereby controlling cell biomechanics. The thesis also investigates the viscoelastic properties of primary articular chondrocytes and the effect of de-differentiation. Micropipette aspiration was used to measure the pressure required for membrane-cortex detachment as well as the apparent equilibrium and instantaneous moduli based on fitting the standard linear solid model to the temporal changes in aspiration length induced by a step negative pressure. Simultaneous live cell confocal imaging of actin dynamics was achieved by transfecting cells with LifeAct-GFP. The studies herein demonstrate that the strength of the membrane-cortex adhesion increased from 0.15kPa in stem cells to 0.71kPa following chondrogenic differentiation of hMSCs. This effect was associated with a reduced susceptibility to mechanical and osmotic bleb formation and an increase in the apparent modulus of the differentiated stem cells as well as reduced cell migration. Differentiated stem cells expressed greater levels of the membrane-cortex ERM (ezrin, radixin and moesin) linker proteins at both gene and protein level. Transfection of undifferentiated stem cells with dominant active ezrin-T567D-GFP increased the strength of the membrane-cortex bond. This suggests that increased expression of ERM in differentiated cells is responsible for the reduced blebability and increased modulus observed. Differentiated cells also exhibited greater F-actin density and slower actin remodelling, based on FRAP analysis of cells transfected with LifeAct-GFP, which may also influence cellular viscoelastic properties. Finally the thesis demonstrates that dedifferentiation of primary chondrocytes also increased F-actin density and expression of ERM linker proteins with associated alterations in membrane-actin adhesion and cellular viscoelastic properties. In summary this study provides new insights into the role of membrane-actin cortex adhesion and the expression of ERM linker proteins in regulating the mechanical properties of chondrocytes and mesenchymal stems cells.

Mechano-regulation of genome function to direct stem cell fate

This project addresses the concept that the nucleus acts as a sensor for mechanical stimuli. By characterising biophysical and epigenetic changes as stem cells differentiate, we will identify pathways responsible for the alteration of cellular mechanosensitivity. These can then be targeted to repair defective mechanosensitivity in diseased or aged cells.

Cell and Tissue Engineering

Articular cartilage, mechanotransduction, cytoskeletal dynamics, calcium signalling, chondrocytes in agarose gel, confocal microscopy.