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

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.

The occurrence of genetic changes in human pluripotent stem cells (hPSCs) has been recognised as one of the major barriers to translation of hPSC-derived therapies. A major concern is a possibility that variant hPSC may compromise the functionality of the final cellular product or render it tumorigenic upon transplantation into a patient. Yet, hPSC are susceptible to acquiring non-random genetic changes (such as gains of chromosomes 1, 12, 17, 20 or X) upon culture. In a large-scale study of the International Stem Cell Initiative (Nature Biotechnology, 2011), as many as 40% of 125 lines surveyed acquired karyotypic abnormalities after 15-20 passages in culture. Genetic variants with recurrent genetic changes display a selective growth advantage, allowing them to rapidly outcompete wild-type cells and achieve clonal dominance in cultures.

To tackle this issue, we will follow up on our recent work suggesting that perturbed mechanosensing confers a selective fitness advantage to variant hPSC. Here, we will combine tools and expertise in genetically variant hPSC (PI, Barbaric) with tools and expertise in mechanobiology of stem cells (co-I, Chalut) in order to determine the role of mechanosensing in variants' fitness advantage and develop conditions that minimise the appearance of genetic variants in hPSC cultures destined for use in regenerative medicine. First, we will ascertain the role of key mechanosensing machinery (i.e. cell-cell and cell-ECM adhesions) in the selective advantage of variants. Further, we will assess how mechanosensing in wild-type and variant hPSC is coupled with signalling pathways that instruct hPSC fate. Finally, we will utilise our previously established matrices (hydrogels) in order to engineer cell culture conditions that reduce selective advantage of variant hPSC. This will involve modulation of hPSC mechanical environment by tuning the hydrogel mechanical stiffness and chemical composition.