Year 1

As an embryo progresses through early stages of development, the position of each cell is precisely defined so that it is provided with the appropriate cues to differentiate into the required cell type. We are just beginning to learn the nature of many of these cues, and it is clear that they include both signals secreted and taken up by cells and also signals sensed by cells as a result of how they are tethered to each other and to their surrounding tissues. While classic cellular responses to cues involve a specific receptor on the cell’s surface responding to a specific extracellular cue, the signals that cells sense as a result of their adhesion properties are more complex. We are working to understand how cells sense and respond to the forces imparted on them by other cells and by their underlying substrate, and how this affects their ability to differentiate into cell types of interest.

We have developed a method to grow human embryonic stem cells on substrates of different stiffness, and have found that this dramatically changes how they organize within colonies and how they then respond to developmental cues. Specifically, when we provide cues to embryonic stem cells to initiate differentiation towards the mesoderm lineage, we find that cells on soft substrates respond much more robustly and differentiate more efficiently than cells on stiff substrates. Mesoderm forms during the developmental process known as gastrulation, and we also see aspects of this complex process recapitulated in our system, specifically when cells are grown in embryo-sized colonies on soft substrates. For example, we observe cells migrate and ingress into a region with similarities to the gastrulation-initiating structure called the primitive streak.

Based on these results, we are designing methods and tools to more comprehensively understand how forces are set up within the differentiating colonies and correlate regions of high or low cell stresses with expression of proteins that are crucial for setting up the cells for efficient differentiation. Using a technique called monolayer stress microscopy, we can monitor how tension fields develop over time in primed and differentiating colonies. We have evidence that cell-cell contacts are strengthened when cells are grown on soft substrates, and that these are required to allow the cells to efficiently differentiate. We have also found regional expression of differentiation markers in the primitive-streak-like regions. We therefore intend to combine these observations to understand how colony organization affects cell stresses, and how these affect subcellular protein localization, which ultimately affects how cells respond to soluble extracellular cues to determine their developmental fate.

This work will ultimately provide us with a better understanding of the fundamental processes by which cells respond to extracellular cues, including how soluble signals synergize with mechanical and tissue-level signals, allowing us to apply these principles to design optimized differentiation systems that mimic the endogenous environments in which cells differentiate during embryogenesis.