Human embryonic stem cells can be changed into virtually any cell type in the adult body. Because of this unique capability, these cells have the potential to cure a vast majority of existing human disorders. Several hurdles exist, however, which need to be overcome before results from the exciting field of stem cell research can be used in the clinic. For example, the factors which govern conversion of stem cells into a variety of tissue types that may find uses in regenerative medicine such as in liver, heart, brain, are not well understood. Our research employs a unique multidisciplinary and collaborative approach to harness the expertise of several leading scientific laboratories to bridge this information gap. In particular, our area of specialization is in understanding how the sugars which coat the surfaces of cells impact processes such as the malignant transformation of cancer cells. The CIRM grant will enable us to apply this same accumulated expertise to study the roles of cell surface sugars in the transformation of human embryonic stem cells into cell types useful for the treatment of human diseases.
Statement of Benefit to California:
Programs funded by CIRM and other state granting agencies will allow California to continue to be at the frontier of stem research for the development of new treatments to cure human diseases. Research such as ours will hopefully enable modern medicine to access exciting new areas such as spinal regeneration, and finding treatments for neurodegenerative disorders for which there is currently little hope for curing. Some illnesses which could be potentially impacted include multiple sclerosis, Alzheimer’s, Parkinson, and Batten diseases. Human embryonic stem cells can be changed into virtually any cell type in the adult body. Several hurdles exist, however, which need to be overcome before results from the exciting field of stem cell research can be used in the clinic. For example, the factors which govern conversion of stem cells into a variety of tissue types that may find uses in regenerative medicine such as the liver, heart, and brain, are not well understood. Our research employs a unique multidisciplinary and collaborative approach to harness the expertise of several leading scientific laboratories in the state of California to bridge this information gap. In particular, our area of specialization is in understanding how the carbohydrates which coat the surfaces of cells impact processes such as the malignant transformation of cancer cells. Carbohydrates densely coat the surfaces of all human cells and as such provide the first contact point for communication of the cell with its environment. For this reason, studies of cell surface carbohydrates have powerfully accelerated medical advances in other fields. We now seek to apply investigations of these carbohydrates to the emerging area of stem cell research. Through initiatives like CIRM, California will continue to lead the nation in the discoveries resulting from collaborative scientific research which will fuel tomorrow’s medical advances.
SYNOPSIS: The goal of this project is to characterize the glycans and glyco-conjugates on the surface of ES cells and on ES-derived cells during neuronal differentiation. The PI proposes to identify the modifications and monitor changes in glycosylation using novel approaches, developed in her lab, for labeling glycoproteins. The approach uses the incorporation of unnatural azide-sugars, which can subsequently be covalently modified using a tagged phosphine probe (via a Staudinger’s ligation) and, captured via the incorporated tag. In Aim 1, the ES cells will be grown, either on feeder layers or feeder free, and labeled by incorporation of the azido-sugars during the course of neural differentiation. This is done in collaboration with Dr. Schaffer at Berkeley. At specific stages, defined cell populations (hESC, EB, Neuro-epithelial, neural progenitors, and dopaminergic neurons) will be isolated by FACS using currently available surface markers. Modified (tagged) proteins will be purified and evaluated by Western blots, and identified using FTICR in Aim 2. In Aim 3, RNA from isolated cell populations will be analyzed by gene chip analysis, and stage-specific expression will be correlated with the production of specific glycans/glycoconjugates. SIGNIFICANCE AND INNOVATION: This proposal addresses highly significant aspects of stem cell research with an in-depth approach to studying glycosylation changes in various stages of cell differentiation. The project is innovative at focusing on the dynamic nature of cell surface modifications, and the Bertozzi laboratory has developed several innovative approaches for addressing the important but difficult issue of glyco-modifications. As the human embryonic cells undergo changes during a culturing process in terms of their morphology and biochemistry, their surface glycoproteins are known to experience significant structural changes. Sugar modifications are already established as essential for regulating key signaling pathways during cell specification and differentiation, but the methods to study this are in their infancy. The cell-to-cell interactions and specific changes during cell maturation are believed to involve glycans as very important recognition factors, so it is essential to learn in detail about structural alterations and the quantitative proportions of such glycan markers. Importantly, this proposal is directed toward the cells that will be cultured into neurons, thus potentially aiding certain efforts in regenerative medicine of the CNS system. While this is by no means the first inquiry into the role of surface glycoproteins in stem cell research, the previous studies in this area were mostly qualitative, using primarily lectins and biological techniques. Consequently, there is a need for new chemical and bioanalytical investigations. This proposed research can precisely fill this existing gap through the use of innovative bioorganic methodologies by the Bertozzi laboratory in conjunction with definitive glycomic and glycoproteomic measurements to be conducted in Lebrilla’s laboratory at UC-Davis. Their combined expertise will be complemented by frequent interactions and the advice of David Schaffer’s group on cell culturing conditions and the protocols needed for the success of this meritorious interdisciplinary project. The project has the potential to identify many novel and useful cell surface markers and to generate a large amount of useful data. It is likely that the proposed research will be feasible during the intended period. The investigators certainly have the necessary expertise, but it is unclear if they have preliminary data of their own on the involvement of glycans in stem cell development. STRENGTHS: The project brings together key expertise in glyco-biology, proteomics, and neural stem cell biology. The project is ambitious but as described is feasible in the time frame to generate a large amount of very useful information. It is high on innovation, and it will likely be essential to carry out these particular experiments in the hESC system, compared for example to the mouse. Dr. Bertozzi (the P.I.) has developed a very useful set of metabolic labeling strategies using sugar azides. Some of these procedures using unnatural sugar substrates and their simple modifications to "visualize" them after metabolic incorporation hold a significant potential in the field of glycobiology. In connection with this proposal, these methodologies can be particularly important to identify the glycoproteins which are newly synthesized over the initial cell stage and during different periods of cell development. The Bertozzi lab has a very unique expertise in this area. The relevant procedures are concisely described and documented through key references. Through collaboration with Dr. Schaffer of the Berkeley Stem Cell Center, there is assurance that the biological materials will be obtained under well-controlled conditions, characterized biologically for their stage-specific status, and isolated prior to perfusion experiments and further chemical and biochemical studies. While some minor optimization may still be required, most procedures and protocols for this seem to be in place. Professor Carlito Lebrilla is a well-known expert in mass spectrometric analysis of glycoconjugates. His expertise will be extremely valuable in providing structural identification quantification of glycan changes associated with different stages of cell differentiation. Moreover, he will be able to assist with other glycoprotein characterization studies. As a sidelight of the proposed investigations, the P.I. and her team want to characterize the genomic transcripts associated with glycosyltransferases, glycan-binding proteins, and other enzymes involving glycoprotein build-up and degradation. These will be using the available technologies (glyco-gene chips) developed by the Consortium for Functional Glycomics. Evaluating expression patterns of mRNA is nowadays fairly routine, so there should be no difficulties in adding this valuable information to what will be otherwise measured on the transforming stem cells. Overall, Dr. Bertozzi is to be congratulated on assembling a talented group of co-workers on a much needed stem cell project. The necessary equipment and facilities of all investigators appear excellent. WEAKNESSES: There are no major weaknesses of this proposal in terms of its scientific merit and qualifications of the investigators. One reviewer felt that it could have been more concisely put together and without some repetitive statements. The only real weakness of the proposal is that the metabolic labeling protocol using the azido-sugars is not yet validated in the hESC system. However, the PI indicates that labeling was achieved in the mESC system, and it seems likely that this expert team will be able to work out the details if they differ. DISCUSSION: This project, though ambitious, is believed to be feasible due to the experience and expertise of the applicant. Although the applicant has not yet demonstrated that the labeling technology proposed to allow characterization of hESC glycans can work in hESC, this is not considered a significant concern given the expertise of the investigator. The importance of looking at hESC glycosylation in a kinetic context during differentiation was emphasized, as was the point that this is what the applicant's lab does. Reviewers noted that federal agencies could easily shy away from what some might perceive as a 'fishing' expedition; however this is an opportunity for new, stage-specific marker identification which can have important ramifications.