Funding opportunities

Detection and Elimination of An Antigenic Non-Human Sialic Acid in Human Embryonic Stem Cells

Funding Type: 
Tools and Technologies I
Grant Number: 
RT1-01141
Funds requested: 
$832 527
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
This application addresses a “significant technical hurdle that needs to be overcome before stem cell research can be effectively translated to the clinic” and falls in the category of “discovery and evaluation of novel tools and technologies”. Proposed work will be performed by researchers at a California-based company and [REDACTED] with the aim of developing diagnostics, tools and possible therapies involving human embryonic stem cells (hESc). The surface of hESc is covered with a dense, complex array of sugar chains. The focus of our work is on a non-human cell surface sugar molecule called N-glycolylneuraminic Acid (Neu5Gc), which is naturally present in large amounts on the cell surfaces and secreted glycoproteins of most animals but not humans. Humans have a genetic mutation that prevents Neu5Gc production, and instead accumulate the precursor form of the molecule N-acetylneuraminic Acid (Neu5Ac). However, human cells can incorporate Neu5Gcfrom external sources. Standard hESc culture methods use animal-derived products and mouse fibroblast feeder layers containing Neu5Gc, which is thus taken up, metabolically processed and expressed on the hESc cell surface-as if it were produced naturally within these cells. The hESc remain contaminated by Neu5Gc even when grown in serum-free culture conditions because most serum replacements also contain animal-derived products. This Neu5Gc contamination of hESc is of significance because all humans havecirculating anti-Neu5Gc antibodies that can initiate in an inflammatory response against this foreign molecule. Therefore, any hESc generated using currently standard conditions would result in Neu5Gc-contaminated grafts, which, if placed into human patients, would be at risk of reaction, or even outright rejection. In light of this very practical concern, the overall goals of the proposed research is to: 1.Show that Neu5Gc-deficient mouse embryonic fibroblasts can function as a feeder layer for growth and maintenance of hESc. The use of these fibroblasts could reduce the contamination and help minimize the risk of rejection. 2. Use a newly developed metabolic technique to eliminate and/or prevent incorporation of Neu5Gc into hESc. 3.Optimize antibody and other techniques for rapid and sensitive detection of Neu5Gc in the quality assurance process of hESc, and hESc-derived biotherapeutics. The technical approaches proposed are: 1.Optimize the use of Neu5Gc-free mouse fibroblasts as feeder layers in human embryonic stem cell cultures. 2.Develop and optimize approaches to eliminate Neu5Gc or prevent incorporation in the first place. 3.Evaluate and optimize use of antibodies and other techniques for sensitive and specific detection of Neu5Gc-containing epitopes. In summary, it is highly likely that safe, effective, unique solutions to Neu5Gc contamination in hESc research will be offered by a California-based enterprise to research and industrial scientists – and eventually to patients.
Statement of Benefit to California: 
Currently, the State of California is unique because it is the only state that uses bond proceeds for its Institute for Regenerative Medicine. The tools and technologies grant awards are meant to provide standardized processes that will overcome current roadblocks, which will in turn assure clinically useful quantities of uncontaminated stem cells in sufficient quantities for the production of stem cell-derived biotherapeutic products. The roadblock that this CIRM Tools and Technologies proposal addressesis the elimination of a non-human contaminant from human stem cells cultured under conventional methods. This is an important innovation that will give the state of California the ability to stay at the forefront of this emerging biomedical market. The expected outcome from the funding of our proposed work would benefit California in many ways. All proprietary technologies and methods used to remove this specific type of hESc contamination will have been discovered or developed in California universities or small California biotechnology companies. The funding of this proposal would also allow California to promote interactions between basic academic researchers and scientists in industry who translate recent discoveries into financially beneficial iomedical products. This by itself would bring esteem to the state as being supportive in innovative technologies necessary at the very basic level of hESc maintenance. In addition, the State of California would continue to benefit financially and biomedically because all the entities named here would remain in California and strive to use vendors and purchase services found in California to keep grant monies in the state. Funding the proposed work for the company,[REDACTED] will also show California as a forward-looking state because it is a woman-owned small business. The already funded principal investigator, [REDACTED], is a California-born Latina, a product of the California education system, and as such would contribute to increasing California-educated California scientists. In short, funding [REDACTED] would increase the number of under-represented minorities in business and graduate-level biomedical scientists in Clifornia. If funded, the proposed work could eliminate a non-human contaminant hESc that has been shown to be harmful because all humans produce antibodies against it and are known to participate in inflammation and possibly the rejection of tissue transplants. California would be unique because it would be the only state in the U.S. to offer the technology to detect Neu5Gc contamination and quickly remove an immunologically significant roadblock for the large-scale production of clinically relevant uncontaminated ESc. We feel that the development of this technology that we have shown to work in on a small scale will allow California to successfully compete, at a time when the U.S. is losing itís competitive edge in the world of stem cell research.
Review Summary: 
This proposal focuses on the issue of contamination of human embryonic stem cells (hESCs) by a non-human sialic acid (SA). Standard hESC culture methods use animal-derived products containing this SA, which is taken up by the cells, metabolically processed and expressed. Humans have complement-fixing IgG antibodies against this specific SA and so transplanting contaminated hESCs poses a risk of rejection by direct complement lysis or activated immune cells. The applicants propose to address this anticipated problem by developing SA-deficient reagents, methods for removing this specific SA from hESCs, and sensitive means for detection of this SA. In the first aim they propose to use SA-deficient feeder cells to grow and maintain Neu5-SA-free hESCs. In the second they propose to test a novel, proprietary compound (Compound X) for efficacy in reducing or removing SA contamination from hESCs. In the third aim they propose to develop multiple techniques for rapid and sensitive detection of the SA. Finally, in the fourth aim they propose to demonstrate that SA-free hESCs are protected from the toxic effects of human antisera containing anti-SA antibodies. Although the reviewers were impressed by the quality of the preliminary data and felt the experiments were feasible, they were not convinced that this proposal addresses a significant roadblock in stem cell biology. The reviewers also found the research team qualified and praised the experienced collaborators enlisted for the project. The reviewers questioned the potential impact of this proposal. They noted that there is still debate within the stem cell community about the importance of contamination of hESCs with this SA. One reviewer suggested that SA levels may decrease over time or number of passages under animal product-free culture conditions, or that there may be some human tolerance for this SA. Others reviewers agreed that the significance of SA contamination must be established definitively before the impact of this proposal can be determined. In addition, reviewers noted that many researchers are working to derive new hESC and induced pluripotent stem cell (iPSC) lines under completely defined, animal product-free conditions. The development of these technologies would seem to make the issue of SA contamination less important. The reviewers found the proposal to be feasible as written. They commented that the preliminary data are strong and support the hypotheses of the proposed aims. However, the reviewers did raise a few issues with the research design. One reviewer noted that, in the first aim, karyotype should be analyzed along with biomarker expression in undifferentiated cells. In addition, SA contamination should be examined as a function of number of cell passages. This reviewer also felt that assessment of embryoid body (EB) differentiation by gene expression analysis is not sufficient and should be confirmed by antibody staining for phenotypic markers of cell types from all three germ lineages. A reviewer noted that the second aim lacks methodological details, such as the concentrations and treatment durations for Compound X as well as how they plan to monitor cell viability. This reviewer also pointed out that applying Compound X to EBs might be much less efficient due to the three-dimensional nature of the cell aggregate. Even small molecules may not reach cells within the inner cells of a large EB due to diffusion transport limitations. In the final aim, the applicants propose to use an in vitro assay to examine the inflammatory response of cells as a function of SA contamination. One reviewer questioned whether this is a valid and predictive model for assessing inflammatory response. Finally, reviewers commented that the proposal contains very little discussion of pitfalls that might be encountered and alternative approaches that could be pursued. The reviewers felt the assembled research team is qualified to carry out the proposed research. They noted that although the principal investigator only recently received his/her PhD, s/he has many years of research experience. The reviewers were enthusiastic about the strong team of collaborators assembled by the PI. They raised a few issues with the budget and commented that while the personnel costs seem reasonable, the supply and subcontract costs seem excessive. Overall, while the reviewers praised this project’s feasibility and the strong research team assembled, they weren’t convinced the proposal addresses a significant roadblock in stem cell research.
Conflicts: 

© 2013 California Institute for Regenerative Medicine