Within the shake of a hand, one can tell that bone is hard, skin is soft, and muscle is in between. The physical properties of each of these tissues are important for their distinct functions in the body. At a much smaller scale, a cell uses its sense of ‘touch’ to determine the physical properties of its surroundings. In this way, cells can discern if they reside in a bone, skin, or muscle microenvironment.
Not only do cells sense the stiffness of their environment, but they also respond to it. The physical properties of the matrix microenvironment direct key cellular decisions including cell division, migration, and cell fate selection. For a stem cell, this information is a valuable cue that instructs it to select a cell fate that matches the physical surroundings. Experimentally, a stem cell becomes a bone cell when grown on a stiff matrix but it becomes a brain cell on a soft one. In the body, a multitude of physical and biochemical cues cooperate to direct intricate cell fate decisions. Relative to well-studied biochemical cues, we are just beginning to understand how cells sense and respond to physical cues. Despite their importance in stem cell biology, we know almost nothing about how physical cues interact with biochemical cues to instruct stem cells to select a specific fate.
Our research seeks to understand how stem cells integrate physical and biochemical cues to select a cell fate. We discovered a specific combination of physical and biochemical cues that, when combined, drive adult human mesenchymal stem cells to select a cartilage cell fate. Culture of these stem cells on a cartilage-like matrix stiffness greatly intensifies their response to biochemical signals to induce cartilage production. By investigating the molecular basis of this response, we have novel insights into the way cells integrate physical and biochemical cues. Building on this foundation, we will uncover new mechanisms by which stem cells sense and respond to their surroundings to select a specific fate. While this research is important for understanding cartilage cell fate selection, these fundamental mechanisms will inform many aspects of stem cell biology, including the maintenance of pluripotency and the selection of multiple cell fates.
Although our primary focus is the discovery of basic cellular mechanisms, our model system has important clinical relevance. More than 6 million Californians suffer from osteoarthritis, a disease characterized by the loss of cartilage physical properties. Understanding the interaction of physical and biochemical cues in cartilage may elucidate the cause of osteoarthritis while advancing new therapies to treat it. Already, our research suggests that the combination of biochemical and physical cues enhances the utility of human mesenchymal stem cells for cartilage repair. The results of our work will be applied to promote development of a stem cell-based therapy for cartilage repair.
Statement of Benefit to California:
This project investigates the cues that direct human mesenchymal stem cells to select a cartilage cell fate. Despite the fact that this stem cell source is very attractive for stem cell-based cartilage repair, several obstacles have limited its clinical application. Our research has identified a novel combination of biochemical and physical cues that overcomes a number of these obstacles. Although we still do not understand how these cues exert their beneficial effects, this strategy has significant therapeutic potential. By investigating the mechanisms by which these cues promote cartilage cell differentiation, this research may advance the translation of these findings to a clinical setting, which would have significant impact on the state of California.
Approximately 6 million adults in California, or 27% of the population have some form of arthritis. This disease costs California nearly $32 billion each year, with an estimated $23.2 billion spent on direct medical care and $8.3 billion due to lost wages. Osteoarthritis is a disabling disease that limits the ability to engage in the regular physical activity that prevents obesity, diabetes, and cardiovascular disease. Consequently, successful development of improved arthritis therapies will benefit the health of a significant portion of the California population.
In addition to the health of Californians, cell-based therapies for arthritis and other musculoskeletal conditions provide a huge commercial opportunity for California industry. Support from Proposition 71 increases the likelihood that such therapies are developed in partnership with California companies. Clearly their economic success will provide employment opportunities for Californians, tax revenue for the state, and help maintain California as a world leader in biotechnology research and development.
Finally, by investigating the cellular response to physical cues, this work has implications for other stem cell based-therapies as well as for biomaterials design. Physical cues that promote a specific cell fate decision can be engineered into novel biomaterials that are used to deliver stem cell-based therapies to any target tissue. Again, these advances have the potential to improve the health of California citizens and to create commercial opportunities for California biotechnology companies.
This proposal is focused on identifying molecular mechanisms underlying the differentiation of human mesenchymal stem cells (hMSCs) into cartilage cells (chondrocytes). The applicant presents preliminary data supporting a synergistic effect of substrate stiffness and a biochemical signaling molecule on chondrocyte differentiation and proposes to study the intracellular signaling pathways downstream of these cues. In Aim 1 the applicant proposes an in-depth analysis of known targets of this signaling molecule, including gain- and loss-of-function experiments as well as evaluation of the roles of cellular localization and post-translational modification. Aim 2 comprises similar experiments to identify targets downstream of substrate stiffness. Finally, in Aim 3, the applicant will evaluate whether the mechanisms of hMSC differentiation into chondrocytes identified through Aims 1&2 in two-dimensional cultures are also at work in traditional three-dimensional hMSC cultures.
Reviewers found the proposal innovative in its focus on the signaling mechanisms underlying mechanosensitive differentiation. However, they felt that the novelty of the experimental system was overstated. Reviewers noted that the substrate basis of this system is not novel and has shortcomings, which are not specifically addressed in the application. The applicant mentions that the preliminary data were generated using a novel cross-linker for the substrate, however the identity of the reagent was not disclosed, making it harder to evaluate the significance of the results. Reviewers did feel that the proposal, if successful, could have a significant impact on the development of therapies for arthritis.
Reviewers raised a number of concerns about the research plan that lead them to question its feasibility. They noted that Aim 1 is well described, but Aims 2 and 3 are less so. Aim 3 is largely disconnected from the first two aims, as there is no clear attempt to control substrate stiffness in the three-dimensional culture model. In addition, the project timeline shows work on Aim 3 starting at the beginning of Year 1, despite its dependence on data obtained in Aims 1 and 2. Reviewers also questioned aspects of the experimental design and would have appreciated additional technical detail. For example, they wondered if the proposed analysis of only two cell lines would be sufficient, considering that shRNA expression can vary widely among stable clones. They also questioned why the non-inductive substrate stiffness proposed in Aim 1 differs from that used to generate the preliminary data. While reviewers found the preliminary data to be generally supportive of the proposal’s hypothesis, they felt it lacked a certain degree of scientific rigor. The reported increases in cartilage cell markers are relatively modest and rely on mRNA analysis, not measurement of protein expression. In addition, use of a positive control in these experiments would have been helpful to demonstrate how close the values are to those obtained from differentiated cartilage cells or cartilage tissue. Finally, not all preliminary data included rigorous statistical analysis, making it difficult to interpret their significance.
Reviewers agreed that the research team is strong, particularly in signaling pathway biology and chondrogenesis. They described the PI and co-investigator as well-published and well-funded, with adequate expertise to carry out the proposed experiments. Reviewers did worry that the lack of technical detail in the proposal could indicate a lack of familiarity with the proposed methods. They appreciated the letter of support from an investigator with extensive knowledge of biomaterials, although it was unclear how the substrate material described in that letter would be used.
Overall, while reviewers appreciated this proposal’s mechanistic focus and strong research team, they raised a number of concerns about the experimental design and preliminary data that led them to question its feasibility.