The role of mechanosensing in the selective advantage of genetically variant human pluripotent stem cells

细胞替代疗法旨在通过替代体内缺失或有缺陷的细胞来治愈疾病或受伤的组织和器官。人类多能干细胞(HPSC)是这类努力的重要支柱，因为它们具有在培养中广泛增殖和分化为任何细胞类型的双重能力。使用hPSC来源的分化细胞治疗黄斑变性、帕金森病和脊髓损伤等疾病和损伤的几项临床试验目前正在进行中，更多的治疗方法也在进入临床试验阶段。细胞替代疗法所需的大量分化细胞的产生通常需要扩增未分化的hPSC并使其长期培养。然而，这一过程的一个关键问题是在长期培养的hPSC中出现非随机的遗传变化(如1、12、17、20或X染色体的获得)。基因变异的hPSC具有反复的遗传变化，与整倍体、野生型细胞竞争，从而在培养中获得克隆优势。这些反复出现的变异显示出肿瘤转化和分化缺陷的迹象，这引发了人们的担忧，即它们可能会对它们在再生医学中的使用构成重大危险。这些变化还可能影响hPSC在其他应用中的使用，包括药物发现、毒理学和疾病建模。因此，解决赋予变异体hPSC适应性优势的机制对于开发旨在将其在hPSC放大过程中的出现最小化以用于研究和再生医学的策略至关重要。我们最近的工作证实，机械传感，即细胞从其环境中感知机械线索的能力，与变异体hPSC的选择性优势有关。在这里，我们的首要目标是研究变异hPSC的扰动机制，以便设计抑制变异超越的培养条件，并允许放大遗传正常的hPSC用于研究和再生医学。首先，我们将描述野生型和变异细胞中的关键机械传感机制(即细胞-细胞和细胞-细胞外基质黏附)，并研究机械传感在变异细胞选择优势中的作用。接下来，我们将研究hPSC中的机械感应如何与指导hPSC命运的信号通路相结合。最后，使用知情的迭代方法，我们将设计降低变种hPSC的选择优势的细胞环境。我们预计，我们的研究成果将包括对变种hPSC的选择优势的机械性理解，以及将变种的出现降至最低的改进培养条件。

Cell replacement therapies aim to heal diseased or injured tissues and organs by replacing the missing or defective cells in the body. Human pluripotent stem cells (hPSC) represent an essential pillar of such efforts due to their dual ability to proliferate extensively in culture and to differentiate to any cell type. Several clinical trials using hPSC-derived differentiated cells for treatment of diseases and injuries, such as macular degeneration, Parkinson's disease and spinal cord injury are currently ongoing, with many more therapies also progressing towards the clinical trials. The generation of large numbers of differentiated cells required for cell replacement therapies typically entails expanding undifferentiated hPSC and keeping them in culture for long periods of time. However, a key issue with this process is the appearance of non-random genetic changes (such as gains of chromosomes 1, 12, 17, 20 or X) in hPSC upon prolonged culture. Genetically variant hPSC harbouring recurrent genetic changes outcompete the euploid, wild-type cells, thereby achieving clonal dominance in cultures. Such recurrent variants show signs of neoplastic transformation and defects in differentiation, raising concerns that they may pose significant dangers for their use in regenerative medicine. These changes may also affect the use of hPSC in other applications including drug discovery, toxicology and disease modelling. Thus, resolving the mechanisms that confer fitness advantage to variant hPSC is pivotal for developing strategies aimed at minimising their appearance during hPSC scale-up for use in research and regenerative medicine.Our recent work established that mechanosensing, i.e. the ability of cells to sense mechanical cues from their environment, is implicated in the selective advantage of variant hPSC. Here, our overarching aim is to investigate the perturbed mechanosensing of variant hPSC in order to design culture conditions that suppress the variant overtake and allow the scale up of genetically normal hPSCs for research and regenerative medicine. First, we will characterise the key mechanosensing machinery (i.e. cell-cell and cell-ECM adhesions) in wild-type and variant cells and investigate the role of mechanosensing in the variant cells' selective advantage. Next, we will study how mechanosensing in hPSC is coupled with signalling pathways that instruct hPSC fates. Finally, using an informed iterative approach, we will engineer cellular environments that reduce the selective advantage of variant hPSC.We expect that the outputs of our research will encompass a mechanistic understanding of variant hPSC's selective advantage and improved culture conditions that minimise the appearance of variants.