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换体内缺失或有缺陷的细胞来治愈患病或受伤的组织和器官。人类多能干细胞（hPSC）代表了这种努力的重要支柱，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衍生的分化细胞治疗疾病和损伤（例如黄斑变性、帕金森病和脊髓损伤）的几项临床试验目前正在进行中，更多的疗法也在向临床试验发展。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并将它们长时间保持在培养物中。然而，该过程的关键问题是在长期培养时hPSC中出现非随机遗传变化（例如染色体1、12、17、20或X的获得）。携带复发性遗传变化的遗传变体hPSC胜过整倍体野生型细胞，从而在培养物中实现克隆优势。这种复发性变异显示出肿瘤转化和分化缺陷的迹象，引起了人们的担忧，即它们可能对它们在再生医学中的使用构成重大危险。这些变化也可能影响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，解决赋予适应性优势的变异hPSC的机制是关键的发展战略，旨在尽量减少他们的外观在hPSC的规模扩大，用于研究和再生medicine. We最近的工作建立，mechanosensing，即细胞的能力，从他们的环境中感知机械线索，是有牵连的变异hPSC的选择优势。在这里，我们的首要目标是研究变异hPSC的扰动机械传感，以设计抑制变异超越的培养条件，并允许扩大遗传正常的hPSC用于研究和再生医学。首先，我们将研究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-ECM粘附），并研究机械传感在变异细胞选择性优势中的作用。接下来，我们将研究hPSC中的机械感测如何与指示hPSC命运的信号传导途径相结合。最后，使用一种知情的迭代方法，我们将设计细胞环境，减少变异hPSC的选择性优势。我们希望我们的研究成果将包括对变异hPSC选择性优势的机制理解，以及改善培养条件，最大限度地减少变异的出现。

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.