Recommended if funds allow
Like a thick frosting on a cake, complex sugar chains decorate every surface of every cell. Try to approach a cell, as friend or foe, and the canopy of sugars is the first gate-keeper. Each cell makes and organizes these sugar chains, called glycans, on its surface. They are very complicated molecules, and different cells choose to decorate themselves with different glycans—for reasons best known to the cells themselves. Because glycans have such complicated structures, it is hard to work with them and understand their function. They are much more diverse than DNA and proteins, and so the technology for dissecting their structures and functions has lagged behind the others in the molecular revolution in biology and medicine. Glycans are complicated molecules, hard to work with, difficult to understand, but they are absolutely indispensable to life. Human genetic disorders where just one step in their assembly is missing causes mental retardation, seizures, blindness and poor motor skills. Glycans are used for communication both within and between cells, and this is especially true when cells signal each other about their past and future journeys within the developing body and exactly where they will go and what they will become during development. Embryonic stem cells have a particular set of glycans on their surface and change as the cells develop into different cell types. What directs these changes? Are they all important? Can we manipulate a cell’s fate or convince it to behave in a certain way by changing—or maintaining—the sugar coating? The scientific literature shows that changing surface sugar chains can have profound effects.New technology in the field of “Glycobiology” makes it possible to analyze minute amounts of material with great precision and define these structures. Thanks to our collaborators, we can produce substantial amounts of human embryonic stem cells that uniformly transition into neural precursor cells. Our plan is to describe in detail these glycan changes as they occur and then determine which are actually essential for cells to reach that point. How do these glycans allow them to go further on to neurons, oligodendrocytes and astrocytes? We hope to exploit these unique sugar signatures to identify and isolate cells that will have a particular developmental fate. This is only the beginning. It is a catalog of events and a parts list, but we know how the parts are assembled and what machines are needed. Since we are only beginning the stem cell enterprise, it’s important to define these elements from the beginning. We hope to use this knowledge to direct and influence stem cells to travel down the paths we prefer, since we already know the path is sugar coated and the coating is essential.
Statement of Benefit to California:
The necessary existence of the CIRM is the largest benefit for the state and demonstrates our commitment to the concept that research and understanding are the keys to a better life for all citizens. By understanding how the NPC’s differentiate and which glycans are important, we may be able to forecast which genes will be likely to cause developmental problems if they contain specific polymorphisms. As individual genome scans for disease susceptibility become more commonplace in the community, we will need to identify those genes. Localized manipulation of the cell surface glycans on injured nerve cells may help stimulate their growth. This has already been seen using injected chondroitinase and sialidase digestions to stimulate robust nerve growth in mechanically injured, debilitated rodents. This specific project benefits the state economically. Success in recognizing specific cell types during the differentiation enables the development of reagents and assay kits to isolate such cells, scaling up the isolation, and adapting the lectin-binding purification concept to other types of differentiating cells. The demand for more therapeutic use of stem cells will require an industry prepared to identify and fractionate those cells with special surface properties, some of those being defined by lectin binding.
SYNOPSIS: This proposal will explore changes in glycans that occur on the surface of human embryonic stem cells (hESC’s) as they differentiate into neural precursors (NPC). They will firstly define the changes that are required for conversion of hESC’s into NPC’s. Secondly, they will define the changes that are required for conversion of hESC’s into NPC’s. Thirdly they will use glycan expression to define differentiation markers. SIGNIFICANCE AND INNOVATION: The approaches described here bring novel strategies to the subject of glycan expression on hESC’s. This is a very innovative proposal and brings an experienced scientist to the field of ESC research. Glycans themselves are very diverse and their functions are many. While there has been much effort in studying gene expression and immunophenotype of hESCs using genomic and proteomic analyses, there has not been much in the way of studying hESC glycomics. This is a relatively novel approach which may provide insight into hESC regulation. Understanding the glycan profile of hESCs will provide us with additional crucial information into an important aspect of stem cell biology. STRENGTHS: The PI is an experienced, productive scientist who is an expert in glycobiology. S/he has important collaborations both within the Burnham Institute and at the University of Georgia. S/he will apply state-of-the-art techniques to the research plan. This is a well-written and thoughtful proposal. The experiments are timely and the approach is novel. The PI is an experienced glycobiologist while Dr. Terskikh seems to have the appropriate expertise in neuronal differentiation of hESCs. The Preliminary Data presented show an indication of possible success in the outlined approach. The PI proposes to first characterize the expressed glycome hESCs and examine the changes as these cells undergo neuronal differentiation. By combining aspects of gene expression, mass spectrometry, and glycomic analysis the PI and his team propose to identify specific genes responsible for changes in glycan structure as the cells differentiate. These glycans and genes can then be used to enrich cell populations for specific fates and perhaps isolate pure hESC subpopulations. WEAKNESSES: As acknowledged, his collaborator, Dr. Terskikh, has had some difficulty in differentiation of NPCs into neurons, astrocytes and oligodendrocytes. This differentiation in vitro is likely to take longer than the three weeks described. The project as proposed is not eligible for NIH funding due to the hESC lines chosen. However, the only rationale the PI provides for using these non-eligible lines is the ability to apply trypsin to the cells. While it is true that the HUES lines from Harvard have been adapted to trypsin passaging, gentle trypsin treatment can be used to passage any hESC line. Therefore, this project could easily be NIH-eligible with a change of hESC line choice. The methodology proposed is very complete. However, there are details pertaining to the analysis which are lacking. For example, there is no mention of analyzing the karyotype of the cells nor is there a discussion of how often and over how many passages the analysis will be conducted. Both the PI and Co-PI only designate 5% of their time to this project. In addition to the two of them the team includes an out-of-state collaborator (0% commitment) and one named and one unnamed (TBD) technician, both at 100% commitment. There is a question as to whether 5% effort from the PI and co-PI is sufficient for this project. DISCUSSION: This is a beautifully written proposal. The approach is thorough, novel, well thought out and timely. The work proposed is ambitious. It was noted that the PI's will commit only 5% of their time to this work. Other minor negatives discussed concerned the relatively weak rationale for using non-federally approved lines, and the details pertaining to the analysis that are lacking.