The ability of human embryonic stem cells (hESCs) and induced pluripotent somatic cells (hiPSCs) to become any type of cell (pluripotency) is controlled, in part, by putting phosphates onto key portions of proteins to activate or inhibit them, a process called phosphorylation. However, most phosphorylated proteins are unknown in stem cells. Proteomics is a cutting-edge tool that has largely been unapplied to stem cells. [REDACTED] has strong expertise in stem cells and in proteomics. We will develop and vlidate novel technologies enabling identification of protein phosphorylation changes that define pluripotency and differentiation to specific cell types in order to advance stem cell applications in basic biology and regenerative medicine. Proteins that are phosphorylated in a specific cell type suggest that they help to specify that cell type. These proteins will be identified and their abundance measured in hESCs/hiPSCs and (as a prototypical lineage) in the neural stem cells (2∫ hNSCs) and more matur nerve cells derived from hESCs/hiPSCs. First, protein fragments (termed peptides) will be separated. Because the peptide mixture will be very complex, the 1st separation will split them into 24 fractions. The 2nd separation, performed separately on each of the first 24 fractions, will split phosphorylated peptides from non-phosphorylated peptides so that each category can be analyzed individually. Phosphorylated peptides and non-phosphorylated peptides will then have their molecular weight measured to hep identify them. Massive quantities of data will be generated. To organize these chemical data for use in understanding biology, a computerized database of proteins and their phosphorylation sites will be developed. This unique database will allow us to discover which groups of proteins work together, thus suggesting key protein functions in stem cell behavior. This database will be freely accessible to all researchers. To demonstrate that the proteins are important in controlling what kind of cells the hSCs/hiPSCs become, protein signaling pathways we have newly identified will be experimentally perturbed and then reconstituted. Activators or inhibitors of proteins in hESC/hiPSC pathways will be used, and the amount of these proteins in the cells will be experimentally increased or decreased to improve the percentage of hESCs/hiPSCs that become hNSCs and nerve cells. These studies will provide insight into which proteins control fundamental stem cell function. One additional benefit will be to minimize ptential problems caused by stem cells that may not mature in the desired fashion and/or could pose a cancer risk when transplanted into patients. Relevant proteins controlling stem cell behavior should be revealed, contributing to rational and safe stem cell-based therapies in the long run. The technology will be applicable to differentiation of stem cells to a broad range of cell types.
Statement of Benefit to California:
The ability to use pluripotent human stem cells ñ whether in the form of embryonic stem cells (hESCs) or as reprogrammed adult cells (called induced pluripotent somatic cells [hiPSCs]) ñ rests on understanding the intracellular signaling that directs their fundamental behavior. The ability of a stem cell to become any type of cell is controlled, in part, by putting phosphate groups onto key portions of proteins to activate them, a process called phosphorylation. However, most phosphorylated proteins areunknown in stem cells. ìProteomicsî is a cutting-edge tool that has largely been, as yet, unapplied to human stem cells but is absolutely key to being able to control stem cell behavior and exploit its potential in research (into developmental disorders, degeneration, injury, and cancer), for drug discovery, or for use directly in cell therapies for a broad range of diseases. [REDACTED] a world leader in stem cells and proteomics, will develop and validate novel technologies enabling identification of rotein phosphorylation changes that define pluripotency and differentiation to specific cell types in order to advance stem cell applications. Massive quantities of proteomic data will be generated and will be organized into a computerized database that carries enormous value to science, the biotechnology and pharmaceutical industry, and medicine. This unique database will allow us to discover which groups of proteins work together, thus suggesting key proteins and their functions in stem cell behavior. A additional benefit will be to anticipate and avoid potential problems caused by stem cells that may not mature in the desired fashion and/or could pose a cancer risk in patients. Relevant proteins controlling stem cell behavior will undoubtedly be revealed, contributing to rational and safe stem cell-based therapies. The technology will be applicable to differentiation of stem cells into a broad range of cell types and for a variety of disease states. The database will be freely accessible to all Califoria researchers and citizens in a rapidly searchable unencumbered manner. The therapies, diagnostics, prognostics, drugs, and drug targets that result from this work (as well as the royalties and fees from the downstream intellectual property generated as a result of this invaluable and unique database resource) will clearly benefit California citizens in terms of both their physiologic and financial well-being. Californians will be the first and preferred beneficiaries at no cost or dramatically reduced cost.