Stem cell pluripotency: Impact of bio-inspired substrates on integrin-dependent signalling and force transmission

多能干细胞（PSC），如胚胎干细胞和诱导多能干细胞，具有在成人体内产生任何细胞类型的潜力。这使得它们成为研究胚胎发育过程中调节特定细胞类型分化的信号传导机制的重要工具，也意味着它们作为基于细胞的再生医学疗法具有巨大的潜力。然而，对于这两种应用，必须在限定的培养条件下繁殖细胞，所述条件通常包含重组人细胞外基质（ECM）蛋白和无血清培养基;使得难以产生研究和医学应用所需的PSC的量。为了解决这个问题，我们最近开发了一种新的分子工程纳米纤维PSC基板。我们已经证明，这种基质可以在延长的培养时间内维持PSC处于多能状态，我们的数据表明，这是由于特异性整合素受体的激活。该项目的目的是确定这些整合素与我们的新型底物的接合所触发的信号通路，并探索这种信号通路如何影响PSC的自我更新和分化。整合素受体感知并调节ECM的机械和生物化学性质以维持PSC自我更新和多能性。整合素依赖性粘附复合物作为与收缩性细胞骨架的物理联系和调节细胞命运的动态信号传导联系发挥作用。不同的整合素表现出不同的生物力学和信号传导特性，直接影响细胞增殖和分化。由于PSC优先使用特定的整合素来接合新的底物，我们的主要假设是：生物启发的纳米纤维触发独特的粘附信号复合物的形成，促进多能性和调节机械力转导。为了验证这一假设，我们将使用多学科的方法，结合蛋白质组学，成像，蛋白质工程和超微结构分析：1）定义在PSC底物上建立的整合素依赖性信号传导网络; 2）评估底物和整合素信号传导对机械力转导的影响; 3）确定整合素信号传导模块对维持多能性的作用。最后，我们将与Cell Guidance Systems合作，评估这种新型底物的商业潜力。

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.