Proteomic Technologies for Systems-Level Assessment of Self-Renewal of Embryonic Stem Cells and differentiation to 2º hNSCs
Tools and Technologies I
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.
This proposal describes the development and validation of novel technologies enabling comprehensive, quantitative proteomic and phosphoproteomic analysis of human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs) and their differentiated progeny. The research team intends to focus on proteomic changes in these cells that support pluripotency and neural differentiation. The first aim of the proposal is to identify and quantify a large number of proteins and phosphoproteins from four different cell populations: hESCs, iPSCs, embryoid bodies (EBs) and neural precursor cells derived from hESCs. Proteins will be identified from cell samples by multidimensional chromatography-tandem mass spectroscopy. The second aim involves the construction of an integrated database to allow cellular pathway analyses based on the proteomic and phosphoproteomic signatures obtained in the first aim for each of the cell populations. The third aim is to utilize and validate these data by gene knockdown and overexpression experiments of predicted key components with the goal of improving neural differentiation of hESCs and iPSCs. The reviewers were enthusiastic about the technology presented in this proposal and believed that its application to stem cell biology would have a significant impact on the field. Clinical applications for stem cells will require strict control of the balance between differentiation and self-renewal in these cells. Yet currently, the signaling pathways that regulate pluripotency and differentiation in hESCs and iPSCs are incompletely understood. While some transcriptome studies have been performed, it is clear that the transcriptome does not always predict the proteome. Moreover, many signaling pathways depend not on protein levels, but on the phosphorylation state of proteins. This proposal addresses these issues systematically. One reviewer, while acknowledging the ambitious nature of the proposal as written, questioned the applicability of data generated from only two hESC lines. This reviewer commented that differences among lines are substantial, and while one can assume that shared phosphoproteins are likely to be meaningful, the group may erroneously discount phosphoproteins not seen in both lines. The reviewer cautioned more strongly about generalizing from data culled from iPSCs, which suffer from donor variability, including effects of donor gender. The reviewers expressed concerns about the feasibility of the proposal, mainly with respect to cell biology. In the first aim the group proposes to generate cell lysates from thirty million cells from each of the four different cell populations. There is no mention of how these cells will be cultured or how the pluripotency of the hESCs and iPSCs will be validated prior to proteomic analysis. It seems impossible that they will be able to maintain pure undifferentiated hESCs when attempting to grow so many cells in bulk culture. The reviewers would have liked to see this challenge addressed in the proposal with supporting data. They stressed that for robust analysis there must be stringent identification of the starting populations or the data will contain a large amount of noise. The group also proposes to generate embryoid bodies (EBs) and subject them to the same analysis but there is no specific methodology in the proposal (the timing of EB formation, medium to be used, etc.). A reviewer commented that the third aim is also weak in methodology, with very little information about how phosphoprotein pathways will be chosen for validation and how targets will be prioritized. The proposal includes a fate-choice map but the reviewer felt this was oversimplified and highly idealized given the complexity of 10k different phosphoproteins. This reviewer also noted a lack of methodology for either knockdown or overexpression approaches. Although it states in the application that these techniques are well established in the principal investigator’s lab, the cited reference appeared to be from another group. Another reviewer commented that the estimate of 15k proteins expressed in hESCs seems very high, given that many genes are expressed at low levels or silenced in bivalent domains, and likely not producing protein at levels detectable using the technologies applied in the proposal. The investigators do not provide expected sensitivities for protein or phosphoprotein detection to evaluate this issue. In terms of the team assembled for this proposal, reviewers were enthusiastic. They commented that the team is a strong, experienced one with plenty of experience in proteomics, bioinformatics and cell biology. Reviewers qualified their doubts about the project’s feasibility by noting that if any team could succeed with this project, it’s this one. One reviewer commented that the budget is personnel driven and quite large. This reviewer felt that the travel request of $12,000 per year is excessive, given that the investigators are all at the same institution. Overall, this proposal describes the application of innovative technology to an important roadblock in stem cell biology. The reviewers praised the research team but raised many concerns about the feasibility of the proposal based on the research design and the ambitious nature of the project.