Stem cell pluripotency: Impact of bio-inspired substrates on integrin-dependent signalling and force transmission
Lead Research Organisation:
University of Liverpool
Department Name: Institute of Translational Medicine
Abstract
Pluripotent stem cells (PSCs), such as embryonic stem cells and induced pluripotent stem cells, have the potential to generate any cell type in the adult body. This makes them important tools for investigating signalling mechanisms that regulate the differentiation of specific cell types during embryo development, and also means that they have huge potential as cell-based regenerative medicine therapies. However, for both of these applications, it is necessary to propagate the cells under defined culture conditions, which typically comprise recombinant human extracellular matrix (ECM) proteins and serum-free media; making it difficult to generate the quantities of PSCs required for research and medical applications. To address this, we have recently developed a novel molecularly-engineered nano-fibre PSC substrate. We have shown that this substrate can maintain PSCs in a pluripotent state over extended culture periods, and our data suggest that this is due to the activation of specific integrin receptors. The aim of this project is to determine the signalling pathways triggered by engagement of these integrins with our novel substrate and to explore how this signalling affects PSC self-renewal and differentiation.
Integrin receptors sense and regulate the mechanical and biochemical properties of the ECM to maintain PSC self-renewal and pluripotency. Integrin-dependent adhesion complexes function as both physical links to the contractile cytoskeleton and dynamic signalling nexuses that regulate cell fate. Different integrins exhibit distinct biomechanical and signalling properties that directly influence cell proliferation and differentiation. As PSCs preferentially use specific integrins to engage the novel substrate, our major hypothesis is: Bio-inspired nanofibres trigger the formation of unique adhesion signalling complexes that promote pluripotency and modulate mechanical force transduction.
To test this hypothesis, we will use a multi-disciplinary approach, incorporating proteomics, imaging, protein engineering and ultrastructural analysis to: 1) Define the integrin-dependent signalling networks established on PSC substrates; 2) Assess the impact of substrates and integrin signalling on mechanical force transduction; 3) Determine the role of integrin signalling modules on maintenance of pluripotency. Finally, in collaboration with Cell Guidance Systems, we will assess the commercial potential of the novel substrate.
Integrin receptors sense and regulate the mechanical and biochemical properties of the ECM to maintain PSC self-renewal and pluripotency. Integrin-dependent adhesion complexes function as both physical links to the contractile cytoskeleton and dynamic signalling nexuses that regulate cell fate. Different integrins exhibit distinct biomechanical and signalling properties that directly influence cell proliferation and differentiation. As PSCs preferentially use specific integrins to engage the novel substrate, our major hypothesis is: Bio-inspired nanofibres trigger the formation of unique adhesion signalling complexes that promote pluripotency and modulate mechanical force transduction.
To test this hypothesis, we will use a multi-disciplinary approach, incorporating proteomics, imaging, protein engineering and ultrastructural analysis to: 1) Define the integrin-dependent signalling networks established on PSC substrates; 2) Assess the impact of substrates and integrin signalling on mechanical force transduction; 3) Determine the role of integrin signalling modules on maintenance of pluripotency. Finally, in collaboration with Cell Guidance Systems, we will assess the commercial potential of the novel substrate.
Organisations
People |
ORCID iD |
| Mark Morgan (Primary Supervisor) | |
| Adams George (Student) |
| Description | The purpose of my project is to determine how cell-matrix interactions regulate stem cell pluripotency. Pluripotent stem cells are characterized by their ability to self-renew and differentiate down multiple lineages (pluripotency). Key to the regulation of pluripotency is a family of cell surface receptors called integrins. Following binding of Integrins to proteins in the extracellular matrix, they recruit a wide-range of kinases, adaptor proteins and cytoskeletal regulators. These components differentially regulate cellular processes such as proliferation and differentiation in an integrin-dependent manner. In my project, I aim to define the integrins and integrin-dependent signaling networks recruited; when pluripotent stem cells engage defined substrates (vitronectin, fibronectin, ZTFN and ZT910) that promote pluripotency and self-renewal. In addition, I aim to determine what effect modulation of cell-matrix stiffness has on specific integrins engaged in the defined substrates and how this regulates stem cell behaviour. Key Findings: Immunofluorescence (IF) of iPSCs plated on vitronectin recruits integrin aVß5, fibronectin recruits a5ß1, ZTFN recruits aVß1 and ZT910 recruits a5ß1 at focal adhesion structures. Adhesion complex enrichment (2DE) was employed to isolate integrin-associated adhesion complexes following adhesion of iPSCs on defined substrates. Subsequent western blot analysis showed enrichment for integrin heterodimers correlating with IF datasets. This was further confirmed through integrin inhibition assays. TFM of iPSCs were cultured on polyacrylamide gels (PAA gels) of different rigidities (soft (5 kPa), medium (10-15 kPa) stiff (70-100 kPa)) to determine if the integrin heterodimers recruited on the defined substrate possess unique mechanical properties. For all substrates, as the rigidity of the substrate increases, the force transmission increased proportionally. Furthermore, significant differences in force transmission were observed between defined substrates at rigidities 5 kPa and 70-100 kPa. To determine how the unique mechanical properties of integrin heterodimers influence stem cell behaviour, I examined YAP localisation and chromatin condensation and organisation. YAP localisation: iPSCs were cultured on polyacrylamide gels (PAA gels) of different rigidities (soft (5 kPa), medium (10-15 kPa) stiff (70-100 kPa)) to determine differences in YAP localisation on the defined substrate. For all substrates, as the rigidity of the substrate increases, no significant differences were observed in YAP localisation contrary to observed differences in literature. Chromatin condensation and organization: iPSCs were cultured on polyacrylamide gels (PAA gels) of different rigidities (soft (5 kPa), medium (10-15 kPa) stiff (70-100 kPa)) to determine differences in chromatin condensation and organisation on the defined substrate. For all substrates, as the rigidity increases no significant differences were observed in chromatin condensation, however, significant differences were observed in chromatin organisation. Proteomic analysis of integrin adhesion complexes formed on defined substrates has identified enrichment of mechanosensitive cytoskeletal proteins described in the "consensus adhesome" in the defined substrates. Furthermore, GO term enrichment analysis has shown that these substrate-dependent differences differentially regulate proteins that modulate the glycolysis and gluconeogenesis. This suggests that the different integrins that are recruited on the different substrates affects the metabolic state of iPSCs. Proteomic analysis revealed that CD98 (SLC3A2) as a candidate protein that may regulated integrin-mediated mechanotransduction and downstream signalling and metabolic state. I am currently conducting knockdown on SLC3A2 to characterise these differences. |
| Exploitation Route | The work carried out in this project will help develop a fundamental understanding of the integrin signalling networks recruited on defined substrates. Furthermore, the force transduction aspect of the project will give us an insight into how these signalling networks are differentially regulated when exposed to different mechanical stimuli. Due to similarities that exist between pluripotent stem cells and cancer stem cells, the knowledge from my research can be translated to better understand how cancer stem cells function maintain primary stem cell populations in vivo. This will open avenues for therapeutics that target mechanisms linked to cancer stem proliferation, survival and metastasis. |
| Sectors | Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |