Human pluripotent stem cells (hPSCs) continue to generate tremendous interest in the scientific community due to their unique ability to differentiate or transform themselves, through a process known as lineage commitment, into any of the specialized cell types found in the human body. These cells may therefore represent a source material for organ and tissue replacement to treat several debilitating ailments ranging from diabetes to Parkinson’s disease. Unfortunately, the current methods for directing stem cell differentiation are notoriously inefficient and difficult to control. Scientists currently lack the tools to properly obtain the large populations of differentiated cells needed for these future clinical applications. For example, traditional cell growth practices and tools rely exclusively on exposing hPSCs to chemicals and biomolecules that influence stem cell behavior. These practices overlook other crucial environmental stimuli such as mechanical signals that are inherently present in native stem cell environments. By ignoring non-chemical environmental factors that contribute to lineage commitment, such as stiffness or modifications to a cell’s internal architecture, cell shape, volume, and mass, conventional culture methodologies often result in unsynchronized growth and differentiation, which has been shown to reduce yields of desired cell types. The work proposed in this project will address this problem by adapting nanotechnology based tools and methodologies, which we originally developed for studying the mechanical properties of cancer cells, to controllably apply well-defined mechanical forces to hPSCs. One objective of our research is to assess the extent to which mechanically stimulating stem cells as they grow can influence the outcome of differentiation. Functionally, these studies will determine how to coax individual stem cells to differentiate to fully functional adult cells. Heart cells will be the initial target for this work based upon their natural tendency to beat in rhythm. Altogether, the nanotechnology proposed here will enable an entirely new approach to culturing hPSCs that will permit scientists to more closely probe the mechanisms behind stem cell differentiation. This research provides the fundamental basis for a technology that has the potential to produce large populations of differentiated cells that will speed the translation of stem cell technologies for therapeutic and diagnostic applications.
Statement of Benefit to California:
There has been tremendous interest in harnessing the unique capability of pluripotent stem cells to both self renew and to differentiate into all specialized cell types found in the human body. The scientific investigation of cells with these capabilities, such as human embryonic stem cells (hESCs) or induced pluripotent stem (iPS) cells, may eventually lead to revolutionary cell based treatments against several debilitating pathologies that affect many citizens of California such as type I diabetes mellitus, Parkinson's disease, and sickle cell anemia. While there is significant promise for this work, the transition from the laboratory bench toward clinically viable therapies has been slowed by several limitations. In particular, current state-of-the-art methodologies for directing stem cell differentiation toward specific lineages are notoriously inefficient and often result in mixed populations of cells. In order for the state of California to maintain its scientific leadership position in the emerging field of stem cell biology and ultimately drive the deployment of clinically relevant stem cell therapies to its citizens and the world, there is a critical need for new tools and strategies that will improve the efficiency and scalability of stem cell differentiation. The work proposed in this project merges concepts from nanoscience, biophysics, materials science, and stem cell biology to develop a means to controllably apply well-defined mechanical forces that will move towards improved differentiation yields. The nanotechnologies described in this proposal enable us to emphasize experimental parameters, which are not attainable by traditional culturing practices, and provide rigorous control over the biophysical microenvironment surrounding the differentiating stem cells. The nanotechnologies developed in this program form the basis for scalable and robust culturing tools that unlock a completely new approach for controlling stem cell behavior. These enabling technologies will not only benefit the scientific community but will also drive emerging commercial and medical development of stem cell therapeutics and other advances that will the benefit the state of California and its citizens.
The application describes the development of a tool that uses a nanotechnology- and imaging-based platform to measure and apply mechanical forces to human pluripotent stem cells (hPSCs) to direct their differentiation. The applicant will first develop quantitative cellular force spectroscopy tools that can facilitate the application of well-defined mechanical stimuli to individual hPSC cells and colonies. Then, the applicant will determine if mechanical force conditions, which manipulate stem cell fate decisions, can be correlated with cytoskeletal signaling and gene expression and can be adapted to promote germ layer differentiation. Finally, the applicant will develop a procedure to guide individual stem or progenitor cells towards a terminally differentiated cell type through the application of mechanical forces.
The reviewers acknowledged the proposed tool’s potential for significant impact, since it might yield a new approach to aid hPSC differentiation. They also found some aspects of the proposal such as the incorporation of a single-cell high-throughput force spectroscopy system into the tool to be highly innovative. However, reviewers criticized the applicant’s suggestions that mechanical forces would provide the most efficient method to facilitate differentiation and that this approach would eliminate the need for chemical (growth factor) supplementation. The absence of compelling preliminary data indicating the particular importance of mechanical forces for cellular differentiation weakened reviewer enthusiasm for the project.
Reviewers felt that the research plan lacked clarity and adequate detail, raising major questions about the feasibility of the proposed research. Reviewers had concerns that the device to apply mechanical forces had not yet been built and tested, and thus, Aims 2 and 3 might not be feasible, since they are incumbent upon the instrument’s development. There were also concerns about the system’s integration with long-term cell culture platforms. Reviewers questioned the biological relevance of the forces applied to the cells, since magnetic beads are significantly smaller than single cells and will only apply localized forces over a small area of the cell. Reviewers were unclear as to how these beads would be physically bound to the cells. Also, the preliminary data did not contain any information about typical stem cell gene expression patterns before or after the mechanical stimulation, and alternative strategies were inadequately discussed. Furthermore, the applicant did not provide sufficient hypotheses as to how mechanical forces might affect cell differentiation, and it was unclear to reviewers that the proposed experimental system would provide the capacity needed to evaluate a wide range of conditions and their effects on cell fate.
The Principal Investigator (PI) has significant experience with force microscopy and is a leading expert in nanotechnology. The research team is comprised of highly accomplished, senior scientists who bring complimentary expertise in stem cell biology and instrumentation to the project. However, junior members of the team are relatively inexperienced.
In summary, this application describes the development of a tool to measure and apply mechanical forces to hPSCs to advance their differentiation program. Although the proposed tool is highly innovative, significant flaws in the project’s rationale, approach, and research plan diminished reviewer enthusiasm. Thus, the application was not recommended for funding.