Tools and Technologies I
There is a critical need for new technologies to facilitate growth and differentiation of human embryonic stem cells (hESC) using clinically acceptable, animal-free reagents. In particular, most currently used culture conditions are not acceptable for standardized production of clinical grade cell products. We propose to develop novel, well-defined, synthetic extracellular matrices for growth and differentiation of hESC. Our approach is to first understand how hESC interact with extracellular matrix materials by analyzing candidate adhesive substrate proteins and integrin receptors that mediate attachment, survival, proliferation and differentiation. Biomimetic, synthetic matrices will be developed, with components and strategies informed by our knowledge of fundamental cell biology. We have established an active, interdisciplinary collaboration between experts in cell biology, stem cell culture, peptide chemistry and materials research. Preliminary data have identified crucial receptors that mediate adhesion and survival of hESC. As proof of concept, novel, biocompatible hydrogel polymers have been developed and analyzed for physical properties, cellular toxicity, and for their ability to support adhesion and growth of hESC. A method for rapid, high throughput screening of candidate hydrogel peptides has been developed using inkjet printing technology. We propose to develop and test peptide-hydrogels for culture of hESC. Peptides from adhesive extracellular matrix proteins will be screened for their ability to support adhesion, survival, proliferation and differentiation of several hESC lines, including some that are not federally approved. Arrays of candidate materials, using single peptides and combinations of peptides will be arrayed using ink jet printing and assayed in adhesion assays. Larger scale experiments will test adhesive substrates for survival, proliferation, and maintenance of the undifferentiated state. The proposed experiments, if successful, will address an important unmet need in bringing stem cell therapies to the clinic and provide the foundation for a wide range of fundamental studies.
Statement of Benefit to California:
The State of California, like the rest of the nation, faces immense challenges to its health care system, with soaring medical costs due in part to continuing care of our aging population. The percentage of elderly in California is expected to grow from what was 14 percent in 1990 to 22 percent in 2030. Chronic degenerative diseases such as Alzheimer’s disease, Parkinson’s disease, age-related macular degeneration, cancer, diabetes, cardiovascular disease, osteoarthritis, and osteoporosis afflict a growing number of individuals in California. Major innovative approaches are now, more than ever, an imperative. Human embryonic stem cells (hESC) have great potential for the treatment of disease and injury because they are pluripotent in their capability to form most cell types in the body. They will also be of great utility for screening new drug candidates, and for understanding the molecular mechanisms of human development and disease. However, methods used to grow hESC are in their infancy, and scale up for production of clinical grade cells will require further research. Our proposed research will develop new methods for culture of hESC using synthetic matrices that will be suitable for clinical applications. If successful, this work will be a great benefit to the state by providing useful new technology that addresses a critical need in the field of stem cell research. In addition, it provide new approaches for therapies to treat degenerative conditions that afflict millions of Californians.
The proposal is based on an interdisciplinary approach to development of defined, robust and scalable culture conditions using synthetic extracellular matrices for undifferentiated hESC expansion. The team is composed of domain experts in cell biology, stem cell cultivation, peptide chemistry and materials science. The group has identified receptors that are mediators of adhesion and survival of hESC. They propose to build on their preliminary data by developing and screening peptide-hydrogels for ability to support adhesion, survival, proliferation and differentiation of cultured hESC. Inkjet printing will be used to array candidate peptides and combinations of peptides for high throughput studies. The candidate peptide-hydrogels identified in the screening assays will be more fully characterized for effects on phenotype and karyotypic stability. The most promising candidate peptide-hydrogels will be further optimized by quantitative examination of mechanical properties that affect particular stem cell outcomes. The proposal addresses the current bottleneck in expanding hESC to numbers and quality sufficient for clinical application (master banks). Current state of the art methods to expand hESC rely on Matrigel as a substrate for cell attachment and trophic support, and many media commonly used in hESC cultivation contain animal products. By focusing on the hESC-extracellular matrix interactions in a systematic way, the proposed work could have enormous impact on methods to control growth and differentiation of hESC to clinically needed numbers. Preliminary data provide solid support for the underlying rationale for the high throughput screening of biomimetic matrices as proposed in the application. The research design follows a systematic and logical approach, though reviewers were not completely sure of the numbers and combinatorics involved in the peptide screening, and exactly how the candidate peptides will be prioritized for further study. Reviewers also appreciated the discussion of potential limitations of the experimental approaches, and alternative strategies. One of the reviewers advised that one of the proposed media components did not fit the goal of examining completely xeno-free methods of hESC expansion since it contains animal-derived product. Since considerable organizational skill is required to coordinate a multidisciplinary team, reviewers applauded the 34% effort of the principal investigator, who is well suited to lead the team of researchers. Each team member is a recognized, respected leader in their particular area of expertise. Thus, with each contributing critical intellectual viewpoints, the lower level of effort of other investigators on the proposal, one of whom appears to be fully committed with other projects, was of some concern. Nonetheless, as a team, this group is exceptionally strong for these kinds of studies. Overall, the application was received enthusiastically for the multidisciplinary approach to an important bottleneck to translation. Though reviewers generally wanted more details of experimental design, they were comfortable that the logical progression of experiments would be feasible and lead to interesting results in the hands of the strong team. Thus potential impact of the work is high.