We have assembled a team of investigators with complementary expertise in applying the state-of-the-art “one-bead-one-compound” (OBOC) combinatorial library methods to identify synthetic chemical molecules that bind to unique receptors (protein molecules) on the surface of human embryonic stem cells and induced pluripotent stem cells. In this technology, stem cells will be mixed with huge number of chemical-beads (1,000,000 or more), and those beads coated by the stem cells will be isolated for chemical analysis. We believe some of the chemical molecules identified by this method will support the growth and proliferation of stem cells while maintaining their “stemness” nature (self-renewal). Other molecules may induce directed-differentiation into specific desirable cell types such as heart cells for damaged heart and brain cells for patients who suffer stroke. Once these molecules are identified, we shall incorporate them into an artificial gel that can support large scale stem cell growth and directed-differentiation. Such artificial gels are free of animal products and viruses, making them safe for therapeutic use in human. We shall take advantage of these novel molecules and gels to study and therefore understand how stem cells work. These molecules may also be used as imaging probes to track or localized stem cells inside patients. For tissue regeneration, we may incorporate one or more of these molecules onto surfaces of biodegradable scaffolding with predetermined shape, so that simple artificial organs with various cell types can be developed. These molecules may also serve as very important tools and reagents for basic stem cell research.
We have assembled a team of investigators with complementary expertise in applying the state-of-the-art “one-bead-one-compound” (OBOC) combinatorial library methods to identify peptide, peptdomimetic, or small molecule ligands that bind to unique receptors on the hESC/iPSC surface. We anticipate that some of these ligands can support the self-renewal and/or pluripotency of hESC/iPSC, while others are capable of inducing specific cell signaling to promote directed differentiation and facilitated maturation of such lineage-specific derivatives as cardiomyocytes. Since D-amino acids, unnatural amino acids, and other small molecule building blocks will be used in the construction of our chemical libraries, the hESC/iPSC-specific ligands identified in this research are expected to be resistant to proteolysis. We envision that these ligands will be invaluable for basic and translational hESC/iPSC research. For instance, when immobilized in a solid support or gel matrix, these ligands (individual or a mixture) could function as an artificial extracellular matrix for promoting self-renewal, pluripotency, (cardiac) differentiation or maturation. Such functionalized scaffold will be useful for tissue engineering, tissue regeneration, stem cell and lineage purification (e.g. cardiomyocytes from a mixture of cell types present in differentiation guided extraction of resident stem cells). High-specificity and high-affinity ligands can also be used for in vivo tracking of stem cells and their derivatives. For example, the ligand can be radiolabeled with 64Cu or 18F, injected i.v. into the patient or experimental subject, and the specific cells recognized will be localized by PET scan. Indeed, we have already successfully used such approach for in vivo imaging of ovarian and lymphoid cancers in xenograft models. Similar to monoclonal antibodies, hESC/iPSC/CM-specific ligands can also be use as reagents in flow cytometry analysis of stem cells.
Many of the technologies that have been or to be developed in this research will not only benefit patients in California, but will also lead to commercialization and establishment of new biotechnology companies in California, and therefore economic growth of our state.
This application describes the identification of small molecule ligands for promoting self-renewal, differentiation, and maturation of stem cells. To achieve these ends, the applicant proposes to use combinatorial chemistry to create peptide libraries that are coupled to beads. This library will be used to screen various pluripotent cell lines that have been lentivirally engineered to express stage and/or cell type specific markers, in order to identify peptides that bind to cell surface receptors on these cells. Finally, the molecules of interest will be comprehensively assessed for their ability to promote stem cell behaviors such as growth, differentiation, and maturation, with a particular emphasis on cardiomyocyte generation. Investigators will use imaging, electrophysiology, proteomic, and microarray techniques to assess cellular responses.
The reviewers were enthusiastic about the proposed technology and its potential advance the field of stem cell biology. The identification of small molecule ligands to manipulate self-renewal, pluripotency and directed differentiation of human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSCs) would be an important advance. Equally, identification of ligands that promote cardiomyocyte differentiation might help in the effort to produce mature cardiac cells from hESCs for cell therapy. Other molecules identified in this screen could serve as markers for stem cells, to be used as imaging probes. Finally, an ability to mimic extracellular matrices might offer new scaffolding possibilities for improved tissue engineering. The potential impact of these studies was considered very high, both for basic research purposes and to advance the field of stem cell biology into the clinic.
In terms of feasibility, reviewers agreed that this proposal would have a high likelihood of success due to the eminent qualifications of the investigators and a “first rate” experimental design. The principal investigator (PI)’s laboratory has created combinatorial libraries that were used successfully in other screens. In preliminary work presented in the application, the applicant identified ligands that promote hESC growth by screening one of these peptide libraries against mouse embryonic stem cells. In addition, the applicant has access to a wide diversity of embryonic cell lines available for this project. Reviewers complained that the research plan lacked experimental details, which impaired their ability to assess the project extensively – for instance, it was not clear how many lines would be tested, whether they would take into account gender differences in the lines, and what the size of the screening library was. One reviewer worried that the use of two peptides on a single bead might result in aggregation, a potential pitfall that was not addressed in the application. Despite these issues, reviewers commented on the proposal’s overall strengths in both concept and methodology, and expressed confidence that the project was feasible given the applicant’s success with previous screens.
The strongest aspect of this application is the research team involved. The applicants were described as highly qualified to conduct the described experiments: the PI is a leader in the field of combinatorial chemistry, with extensive experience in the proposed methodologies, whereas the co-investigator provides valuable and complementary expertise with stem cells, cardiac differentiation and lentiviral delivery.
Overall, this is an ambitious and feasible proposal from a talented team of investigators that could potentially advance the field of stem cell science.