Theme
To understand and modify at the nanoscale force- and geometry-sensing pathways in health and disease.
Our Approach
What does mesenchymal stem cell differentiation have in common with immune synapse formation, the foreign body response and the generation of fibrosis? They are all mechanically dependent processes that rely upon the nanometer-level force sensing protein modules in cells and the response mechanisms that are activated in those cells. There is recent evidence that similar cellular mechano-sensing mechanisms underlie these diverse activities but the sensory output is processed differently to activate different modular motile functions. The Nanotechnology Center for Mechanics in Regenerative Medicine has developed nano-tools and expertise to measure the pattern of force and rigidity sensing to enable us to generate new treatments or drugs for directing cell growth and differentiation. In the short term, we will apply these tools to the development of new ways to improve the expansion of immune cells for immunotherapy and more generally improve methods for expanding and differentiating stem cell populations.
Medical Significance of Mechanical Factors
Recent publications and meetings have highlighted the importance of a finely-tuned combination of physical and biochemical factors in controlling tissue regeneration, immune function, cancer progression and a variety of diseases. In cancer, for example, there is evidence that the metastatic seed cells have a greater probability of growing in the right tissue environment, and that physical factors co-regulate their proliferation and differentiation. A similar impact of physical factors has been described for stem cells and is found for immune function as well. An integrated interdisciplinary approach is urgently needed to understand how physical factors are sensed and produce appropriate responses over time to develop (regenerate) and maintain the proper tissue function. The read-out of physical factors by cells involves alterations of biochemical signaling responses. We have described several molecular force transduction mechanisms but we need additional tools (physical and biochemical) to determine which mechanisms are responsible for functions at the cellular level. To measure and generate forces at the specific molecular-scale locations during these functions, a number of nano-structured devices were designed and built by a new high throughput process. Our previous biochemical studies and those of other labs have identified potential target molecules that are responsible for the mechanotransduction in immune and stem cells. These can be combined with tools for making matrices of different rigidities and strains to screen for the desired effects on specific cell functions in the immune and mesenchymal stem cells.
| Steps In Mechanical Signaling Pathways |
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At the basic science level, a number of important findings have fundamentally altered the way that we approach biomechanical problems. First, there is a growing consensus that similar mechanical signaling pathways play important if not critical roles in immune and stem cell functions. We have found that both cell types mechanically pull on ligands and matrices and the mechanical resistance that they encounter plays a critical role in the signal that is generated. The immune synapse involves the motor-dependent, mechanical sorting of the t-cell receptors and other components. Further, mechanical aspects of the ligands control the t-cell response. Similarly, mesenchymal stem cell growth and differentiation is strongly dependent upon the mechanical characteristics (geometry and rigidity) of the matrix. Another similarity that is emerging is that the forces exerted by the cells will alter the environment. Immune cells will perturb the presenting cells by pulling on them and stem cells will stretch matrix that can then alter matrix chemical and physical properties. Molecular-level studies show that tyrosine kinase phosphorylation of stretched proteins or protein binding to stretched-exposed sites can provide a primary cue for force-dependent signaling and important proteins in immune cells and stem cells are likely candidates to be stretched. Thus, the study of the mechanisms of mechanosensing in the two systems is complimentary and each enlightens the findings of the other.
A critical aspect for the Center is the development of new nanometer level devices for measuring or creating sub-cellular forces. Nanoimprint lithography (NIL) has proven to be a reliable way to fabricate samples in far greater quantities (and far less time) than would be possible by direct-write electron beam lithography. All members of our nanofabrication team incorporate NIL into their fabrication schemes, and we have developed some new techniques and optimized others, making our NIL fabrication more robust and repeatable. At multiple scales from sub-10 nm dimensions to 10 micrometers, we can stamp out surfaces with NIL. In addition, we have developed new techniques for fabricating elastomeric surfaces with multi- and variable rigidity with micron-scale and nanometer-scale precision. The next generation of devices is being fabricated to enable periodic stretching of the surface to mimic in vivo activity of tissues. These devices are critical to understanding the roles of geometry and force in cell function and behavior and they are designed to be generally applicable to immune and mesenchymal stem cells.
Our Pathway to Medicine
Our Pathway to Medicine is growing rapidly and we have important new initiatives to apply the expertise and the tools from the analyses of biomechanical processes to the development of treatment protocols. Closest to the clinic is an initiative headed by Michael Milone (UPenn) that is developing new surface patterns for the amplification of memory t-cells from patients for immuno-therapies. Preliminary findings with Michael Dustin and Lance Kam show that the spatial patterning of adhesion sites can cause dramatic increases in t-cell proliferation and commitment to different differentiation pathways. Our effort on stem cells is more general and involves the development of new surface features in a systematic manner to stimulate specific growth or differentiation patterns in combination with hormone treatments.
Integration of nanoscale technologies, combined with cell biology and computational modeling has provided new quantitative insights into mechanical regulation of cell function. These advances were made possible by the interaction of Center members working toward the common goal of understanding important aspects of cellular mechanics at the nanometer scale. The tools that have been created are now ready to be applied to the engineering of specialized environments for directing growth, differentiation and fate of immune cells and stem cells. The Nanotechnology Center for Mechanics in Regenerative Medicine has begun to apply these technologies and has plans for many more applications that are being tested. Such a coordinated analysis of the physical parameters in conjunction with the biochemical changes has only been possible through such a center, where the many aspects can be approached in parallel and different types of expertise can be applied to the same cell systems simultaneously. Presentation of the signals in physically different patterns changes the cell response and we plan to develop the proper pattern for the appropriate signal generation leading to regenerative or replacement cells.