Recombinant Ab markers for stem cell differentiation

DESCRIPTION (provided by applicant): The potential of stem cell therapies to benefit patients afflicted by some of the most pervasive degenerative diseases of an aging population have made this an intense area of research focus. There is a corresponding need for reagents that permit the molecular characterization of stem cells and their differentiated progeny. Here human embryonic stem cell lines which are genetically marked by incorporation of a fluorescent reporter protein into cell lineage specific genes will be used as bait for recombinant phage antibodies that specifically interact with cells progressing along the marked lineage. A positive-negative selection by FACS will be employed allowing repeated enrichment steps to achieve the isolation of highly specific reagents. Mass spectroscopy will be used as a primary approach to identifying the cognate antigens. Reagents will be additionally characterized for their utility in ELISA, Western blotting and fluorescence in situ detection on fixed cells. Two Phase 1 goals are identified: Goal 1. Generation of antibody reagents allowing detection of specific cell lineages during human embryonic stem cell differentiation. This will be accomplished by the selection of specific binders from a high diversity scFv phage display library at unique differentiation stages by taking advantage of fluorescent reporter marked gene trap cell lines and fluorescence activated cell sorting. Goal 2. Characterization of antibody reagents identified in Goal 1 to define the target antigen and characterize the binding properties of the antibodies. Target identification will be accomplished by immunoaffinity enrichment of the target antigen combined with MS/MS mass spectroscopy and confirmed by standard biochemical techniques such as Western blotting and immune precipitation. (The NIH Human Embryonic Stem Cell Registry cell line UC06 will be used in the proposed work.)