Mechanisms of mechanical regulation of stem cell fate

干细胞命运的机械调控机制

• 批准号: BB/V001140/1
• 负责人: Martin Humphries
• 金额: $ 72.15万
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FV001140%2F1
• 关键词: Mechanisms; mechanical; regulation; stem; cell

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.

机械生物学的新兴领域已经确定，细胞行为受到周围环境的机械特性的影响。这种影响的一个关键结果是细胞命运可以由微环境指导：例如，当在软材料上培养时，人类间充质干细胞（huMSC）采用成脂谱系（即形成脂肪组织），但在硬材料上是成骨的（即形成骨组织）。huMSCs的良好表征的刚度敏感性对于再生医学具有优势，其中通过机械刺激指导谱系的能力引发了对组织工程潜在应用的研究。然而，细胞机械传感的原理具有更普遍和基本的生物学重要性：在胚胎发生和发育中，组织必须保持稳定以使它们能够稳健地发挥功能;在健康组织中，必须保持机械稳态;在衰老和纤维化等疾病中，机械稳态的破坏可能导致病理学。力从微环境传递到细胞（反之亦然）通过嵌入细胞膜中的整合素粘附复合物（IAC）。这些复合物与细胞外基质（ECM）中的蛋白质结合，并与细胞骨架的结构蛋白相互作用。在细胞内，细胞骨架又通过LINC（核骨架和细胞骨架的连接体）复合物连接到细胞核，LINC复合物跨越核膜并通过核纤层附着到染色质。因此，在微环境和转录调控的细胞中心之间存在机械连接的连续管道。将机械刺激转化为生化信号传导的过程称为机械转导。随着机械生物学领域的建立，已经确定了许多关键的机械转导途径;然而，这些通常是通过单个蛋白质实体的活性来确定的。在这里，我们提出了一个系统的和综合的调查，在整个机械传递途径的蛋白质相互作用，适用于检查人骨髓间充质干细胞，因为它们响应环境刚度的变化。假设主导和综合的全球方法相结合，将使我们能够解决以下目标：（a）使用邻近标记方法，结合质谱蛋白质组学，我们将产生一个与细胞机械传递途径（包括IAC和LINC复合物）的每个组成部分相互作用的蛋白质目录。(b)然后，我们将量化蛋白质与细胞机械传递途径的组成部分的相互作用如何在软，中间和硬材料上培养的huMSCs中改变。这将使我们能够建立一个参与机械传感的蛋白质相互作用的候选列表。(c)我们将通过引入突变来去除它们或抑制它们的活性来测试所鉴定的蛋白质相互作用的重要性。力学敏感性受损的后果将通过确定huMSCs是否仍然能够进行adipo和成骨谱系的刚性定向承诺来评估。我们希望通过该项目取得的进展不仅将作为更详细研究细胞微环境和基因表达之间联系的基础，而且还将使得能够开发出在组织工程和治疗应用中控制细胞行为的策略。