Being pluripotent, human embryonic stem cells (hESCs) have the potential to act as a source of cells for regenerative therapies. But before these potential applications of hESCs can be effectively pursued, an understanding of the complex cellular relationships existing within hESC cultures and factors involved in the maintenance of hESC properties is required. hESC lines are known to be morphologically and phenotypically heterogeneous. We propose to select an antibody diversity library directly on live hESCs to identify a panel of monoclonal antibodies that recognize distinct hESC subpopulations, and to further characterize these subpopulations with regards to pluripotency and capacity for self-renewal. These antibodies will be of broad interest to laboratories that are either utilizing and optimizing the existing hESC lines or developing new hECS lines. These antibodies can be used in the future to ensure quality of hECS cultures, develop sublines that are more suitable for regenerative therapy, and identify corresponding antigens for functional studies of hESCs.
Statement of Benefit to California:
This study aims to develop antibodies that recognize distinct subpopulations of human embryonic stem cells (hESCs), and to further characterize these subpopulations with regards to pluripotency and capacity for self-renewal. These antibodies will be of broad interest to laboratories that are utilizing or optimizing existing hESC lines, or developing new hESC lines. This study benefits California and its citizens because the knowledge and reagents generated can be used in the future to ensure quality of hESC cultures, and develop sublines that are more suitable for regenerative therapy.
SYNOPSIS: In this proposal the PI will utilize phage display to identify novel antigens expressed on the surface of hESCs. The overall goal is to identify markers that allow better definitions of functionally pluripotent sub-populations, and to provide novel reagents to the hESC community. Further studies are proposed to characterize that antigens identified by the phage display procedure. SIGNIFICANCE AND INNOVATION: Currently there is some confusion over the homogeneity or heterogeneity of hESC lines. The ability to define markers suitable for analyses of viable hESCs would have an important impact on the field. Phage display is one attractive option to accomplish this goal. Overall, the use phage display top identify markers on the surface of hESCs is innovative. The significance of the proposed studies is high. It is a worthy goal to develop new antibodies that distinguish hESCs from their differentiated progeny, and that increase the precision with which the differentiation of these cells can be characterized. The approaches proposed for doing so are also innovative, relying on sophisticated phage display approaches. However, it is hard to predict the impact that this work will have as it is uncertain whether this work will lead to valuable new reagents. For example, the applicant describes substantial efforts to identify new antigens on cancer cells using phage display. However, this work does not seem to have led to important new insights into cancer biology or treatment yet. STRENGTHS: The major strengths of this proposal lie in the PI's prior success in applying broad phage display single chain antibody technologies to identify antigens expressed on viable cells. The PI proposes a reasonable approach for the identification of new antibodies against informative surface antigens on hESCs. This is an elegant technique that is quite difficult, and the preliminary results obtained in tumor systems are encouraging. Clearly, this is one laboratory where such studies are likely to succeed. WEAKNESSES: The major weakness on this proposal is the difficulties that will be encountered when attempting to functionally analyze hESC sub-populations at the single cell level. This ability is critical for the success of the proposed studies, and is currently problematic for many laboratories. Nevertheless, there is enthusiasm for these studies, given the value of potentially novel antibody reagents for the hESC field. Although the PI is clearly very skilled and focussed on the application of phage display to the discovery of informative new antigens and useful new antibodies, it is not clear whether his/her substantial work in this area has yet paid off in terms of new insights into the biology or impactful new reagents. Thus, it remains to be determined whether the proposed approaches are well suited to the generation of high impact results. The PI was very successful as a graduate student, but the productivity and impact of his/her work as a postdoc and as an assistant professor have been lower. The proposal should place more emphasis on the value of new antigens expressed on differentiated cells as well. Markers of differentiated cells can be as valuable as hESC markers to exclude these cells from hESC preparations and to better identify specific types of derivatives. Differentiation markers will emerge from the proposed experiments, but the screening experiments (while focussed) are described in such a way as to suggest that potentially valuable differentiation markers will be ignored. Some of the methods were described in terms that made it difficult to understand the approaches that were being taken. DISCUSSION: It is difficult to predict the significance of the interesting work in this proposal. The aim is good, the approach is interesting, and there doesn't appear to be anyone doing similar work (applying phage display technology to generation of antibodies to hESC antigens), but prior data/results are lacking. There is a major issue regarding the low productivity of the PI who has other funding but has not produced results. The PI certainly may find new markers for hESC differentiation states, but studies appear to have been on-going for several years in cancer cells and not much has been found/published. In summary, there was concern regarding validity/success of this approach in general as well as success of applicant in applying this technology to generate new reagents.