Statement of Benefit to California:
Year 1The goal of our research is to understand the role of Eph/ephrin signaling in the proliferation or differentiation of human embryonic stem cells (hESCs). Ephs and ephrins are cell contact dependent signaling molecules and have been shown to be important for stem cell differentiation decisions during the development of many structures in the mouse, including the brain, pancreas, and intestine. Our hypothesis is that these molecules will also be used in many differentiation decisions in the human. Additionally, because these proteins are on the cell’s surface they can be used to purifiy specific cell types. In the last year we have differentiated hESC into neurons, as assessed by morphological characterization and expression of marker proteins. We have found that multiple members of the Eph/ephrin signaling family change expression patterns during neuronal differentiation. We have made polyclonal antibodies against one of these, and found that it is expressed in subsets of hESCs. Our hypothesis is that this protein is important for cell-cell contact dependent signaling within a stem cell colony that is used to keep cells in the stem fate. We are currently testing this hypothesis by treating hESCs with agonists of ephrin signaling and assaying for changes in differentiation.
Year 2In the past year we have made important progress towards understanding the role of the Ephs and ephrins classes of signaling molecules in the regulation of hESC growth and differentiation by creating novel tools to detect their expression. These families are large and often multiple members are expressed together during the development of many tissues in the developing mouse. We have determined that many of the 22 members of this large gene family are expressed in human embryonic stem cells hESCs and found that some change their expression upon differentiation of hESCs into human neurons. This leads to the idea that there will be an Eph/ephrin code that can be used to sort and purify specific cell types. We have made several anti-human Eph and ephrin antibodies that recognize the human protein. We have used these antibodies to localize the protein in human stem cells and their differentiated counterparts and determine which types of cells they are expressed in using co-localization with marker proteins. These antibodies will allow us to further probe the function of these proteins. We have perturbed Eph and ephrin function in hESCs using agonists and not detected any difference in cell behavior upon differentiation.