Regenerative therapies require effective differentiation of stem cells to cell types that are functionally identical to those found in vivo. Many current differentiation protocols merely involve optimization of proteins added to the culture media, but do not consider the microenvironmental context in which cells differentiate during development or tissue repair. When we include the biophysical parameter of substrate stiffness, we are able to enhance differentiation of human embryonic stem cells to multipotent mesodermal progenitors, cells that can go on to form muscles, cartilage, and bone. We observe that this differentiation is accompanied by colony-wide organization and coordinated movements. Mesoderm forms during the developmental process known as gastrulation, and we also see aspects of this complex process recapitulated in our system. For example, we observe cells migrate and ingress into a region with similarities to the gastrulation-initiating structure called the primitive streak. We can therefore use this system to optimize directed differentiation protocols, to characterize and manipulate the forces and mechanisms required for coordinated differentiation, and to identify signals involved in primitive streak formation. Together, these studies will allows us to answer questions about the signals required for cell type specification and migration during spontaneous self-organization in the developing embryo.
Regenerative medicine requires efficient generation of cells that are identical to their respective population in the human body, but traditional protocols often lead to inefficient or incomplete directed differentiation. By optimizing biophysical parameters such as substrate stiffness and colony geometry, we show that we are able to efficiently differentiate human embryonic stem cells to mesoderm progenitors, indicating that we can use engineering principles to design more efficient directed differentiation strategies. In addition to providing tunable parameters for any type of differentiation, this system also allows us to probe the molecular basis of early mesoderm differentiation. This will enable us to gain valuable insight into how physical parameters regulate this process in vivo, which is crucial for establishing robust tissue regeneration techniques. In our system, mesoderm commitment is accompanied by cell movements and colony-wide organization representative of some aspects of early embryogenesis, which we will study in more detail to understand how biophysical forces initiate and reinforce key signaling pathways required during morphogenesis. By tracking and manipulating cells during these gastrulation-like movements, we will be able to identify relevant proteins and knock them down to mimic disease states with the ultimate goal of rectifying human embryologic defects and informing the future of regenerative medicine.
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.