Using biomaterials to investigate how ageing affects the anti-inflammatory properties of MSCs and their EVs

随着人口日益老龄化和慢性病的流行，保持更长时间的健康已成为一项严峻的挑战。随着我们年龄的增长，我们的免疫系统发生变化，导致炎症反应增加，使我们更容易生病和受伤；这个过程被称为炎症。需要推行有助于更健康老龄化的新治疗战略。使用间充质干细胞(MSC)，这种细胞遍布全身，有助于正常的组织功能，正在试验作为一种基于细胞的疗法来治疗与年龄相关的疾病。重要的是，MSCs既可以调节免疫系统，减少炎症，又可以促进组织再生。MSCs通过细胞间的相互作用、抗炎酶和可溶性因子的分泌以及来自MSC的颗粒(称为细胞外小泡(EV))来操纵免疫系统。EV含有与它们的亲本MSC相似的蛋白质、DNA和脂质，这使得它们也可以操纵免疫系统的细胞。未分化、幼稚的MSCs已被证明是最有效的治疗方法，因为它们具有最强的抗炎特性。然而，MSCs在体内是一种罕见的细胞群体，因此需要在实验室进行扩增，因为大量的这种细胞被用于有效的治疗。不幸的是，培养条件影响MSCs，导致其抗炎和组织再生特性的丧失，导致随着时间的推移分化为成熟的基质细胞，失去其治疗效果。这种功能的变化受到多种因素的影响，例如它们周围的环境和它们生长的表面。例如，在工程化生长表面培养的MSCs可以影响MSC的新陈代谢，并在培养过程中更长时间保持其功能性抗炎特性。此外，供者年龄和组织来源都影响MSC的功能特性；较年轻的供者MSCs在输入患者时比较年长的供者细胞具有更强大的功能治疗特性。因此依赖于较年轻的捐赠者MSCs，从而减少了可以用这种有效疗法治疗的潜在患者的数量。老龄化人口将从用于细胞治疗的MSCs的替代来源中受益更多，寻找新的方法在实验室扩大功能、临床有效的抗炎幼稚MSCs仍然是研究的关键挑战，减少对年轻捐赠者的依赖。本项目的目的是调查通过调节线粒体功能来改变MSC新陈代谢是否可以帮助老年捐赠者MSCs在实验室扩张时恢复活力。能量的产生与MSC的功能密切相关，本研究将探讨不同培养条件下老年和年轻供体MSCs之间线粒体功能和分布的变化。具体地说，这与MSC和分泌的EV抗炎特性和衰老有何联系。为了评估抗炎作用，将测量MSCs和EVS对免疫细胞活性的抑制。将使用细胞老化的特征标记物来研究老化对扩张期间线粒体功能的影响。当MSCs在特定的生物材料生长基质上扩增时，还将评估其对线粒体活性的影响，以了解细胞与其生长表面的相互作用如何调节细胞代谢，以促进老年捐赠者MSCs的年轻化。由于MSCs功能特性的已知变异性问题，所有实验都将在多个捐赠者身上重复进行。对衰老如何影响细胞过程的深入了解将有助于为扩大MSCs进行基于细胞的治疗创造更好的生长条件。

With a progressively ageing population and increasing prevalence of chronic diseases, remaining healthier for longer has become a heightened challenge. As we age our immune system changes causing an increase in inflammatory responses, making us more prone to disease and injury; this process is termed inflammaging. New treatment strategies which contribute towards healthier ageing need to be pursued. Using mesenchymal stem cells (MSC), which are found throughout the body and aid with normal tissue function, are being trialled as a cell based therapy for treating age-related diseases. Importantly, MSCs can both modulate the immune system, reducing inflammation as well as promoting tissue regeneration. MSCs manipulate the immune system using cell-cell interactions, secretion of anti-inflammatory enzymes and soluble factors, as well as particles derived from the MSC, termed extracellular vesicles (EV). EVs contain similar proteins, DNA and lipids to their parent MSC allowing them to also manipulate cells of the immune system. Undifferentiated, naïve MSCs have been shown to be most effective for therapy as they have the strongest anti-inflammatory properties. However, MSCs are a rare population of cells in the body, so expansion is in the laboratory is required as large numbers of these cells are used for effective therapy. Unfortunately, culture conditions affect MSCs resulting in loss of their anti-inflammatory and tissue regenerative properties resulting in differentiation into mature stromal cells over time, losing their therapeutic efficacy. This change in function is affected by multiple factors such as their surrounding environment and the surfaces they are grown on. For example, MSCs cultured on engineered growth surfaces can influence MSC metabolism and maintain their functional anti-inflammatory properties for longer periods during culture. Additionally, donor age and tissue source all affect MSC functional properties; younger donor MSCs have more potent functional therapeutic properties compared to older donor cells when transfused into patients. Thus there is a reliance on younger donor MSCs, reducing the number of potential patients who can be treated with this effective therapy. The ageing population would benefit more from alternative sources of MSCs for cell therapy, and finding new ways of expanding functional, clinically effective anti-inflammatory naïve MSCs in the laboratory remains a key challenge for research, reducing the reliance on younger donors.The aim of this project is to investigate if altering MSC metabolism through modulating mitochondrial function can aid the rejuvenation of older donor MSCs during expansion in the laboratory. Energy production is closely linked to MSC function, and this study will investigate how mitochondrial function and distribution is altered between older and younger donor MSCs during different culture conditions. Specifically, how this is linked to MSC and secreted EV anti-inflammatory properties and ageing. To evaluate effects on anti-inflammatory properties MSCs and EVs suppression of immune cells activity will be measured. The effects of ageing on mitochondria function during expansion will be investigated using characterised markers of cellular ageing. Effects on mitochondrial activity will also be assessed when MSCs are expanded on specific biomaterials growth substrates to look at how the interaction of the cell with its growth surface can modulate cell metabolism to promote rejuvenation in older donor MSCs. All experiments will be repeated with multiple donors due to known variability issues with MSCs functional properties.A deeper understanding of how ageing impacts cellular processes will contribute towards better growth conditions for expanding MSCs for cell-based therapies.