The applicants' recent derivation of NHP (rhesus monkey and common marmoset) and human ES cells has widespread implications for human developmental biology, drug discovery, drug testing, and transplantation medicine. The requirement by primate ES cells for a fibroblast feeder layer is the most important limiting factor in the routine large-scale expansion of NHP ES cells, and identifying the specific fibroblast-produce factor(s) that mediate NHP ES cell self-renewal is a critical research need. The applicants' preliminary results demonstrate that the essential factor(s) produced by fibroblasts are not secreted, and thus are either membrane bound or are tightly attached to the cell extracellular matrix. Functional strategies for identifying the essential signaling molecules produced by the fibroblasts are hampered by the fact that the molecules are not secreted; therefore novel approaches are required. The applicants' underlying hypothesis is that the ligands and receptors mediating NHP ES cell self-renewal are fibroblast and ES cell integral membrane proteins. To identify these essential signaling molecules, the investigators will accomplish the following specific aims: 1) They will use phage T7 display of fibroblast cDNAs to screen for fibroblast-produced polypeptides that specifically bind rhesus ES cell surface molecules. Polypeptides that specifically bind rhesus ES cells will be quantitatively assayed for stimulatory effects on rhesus ES cell self-renewal. Cloned fibroblast- produced polypeptides will be used to identify the ES cell surface receptor to which they bind. 2) The investigators will use a phagemid (M13) library to isolate high affinity single chain Fv antibodies that specifically bind to the cell surface of rhesus ES cells. Antibodies that bind rhesus ES cells will be quantitatively assayed for inhibitory effects on rhesus ES cell self-renewal. Antibodies that act as antagonists will be used to identify the ES cell surface receptors to which they bind.
