Recommended if funds allow
Statement of Benefit to California:
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.