An important aspect of understanding stem cell biology is to have a basic understanding of the processes that balance stem cell self-renewal and differentiation. Stem cell proliferation and differentiation signals are at least partially regulated by direct contact between cells. For example, stem cells normally reside in a specific microenvironment, or “niche”, that integrates specific cell-cell contacts in order to translate information from the environment into proliferation patterns. In this SEED proposal we plan to investigate the role of Eph/ephrin signaling in hESC growth and differentiation. Eph receptor tyrosine kinases and their ligands, ephrins, are large gene families that initiate signal transduction pathways which lead to changes in cellular adhesion, proliferation, and migration. Both Ephs and ephrins are expressed on the surfaces of cells, thus restricting their interactions to sites of direct cell-cell contact. It is known that Ephs and ephrins are expressed in hESCs and are therefore in the right place to be involved in regulating hESC proliferation and differentiation decisions. To better understand how Ephs and ephrins might be involved in hESC growth regulation, we plan to characterize the expression of Ephs and ephrins in hESCs during different stages of growth and neural differentiation to determine if Eph/ephrin signaling is used to regulate proliferation and differentiation of hESCs. The characterization of the role that Ephs and ephrins play in hESCs will provide insights into how stem cell proliferation is regulated in culture and will likely be applicable to how stem cell niches are organized in vivo. This understanding may allow for the development of standard culture conditions that will optimize both self-renewal and homogeneity of cells. This will in turn lead to more efficient large-scale production of stem cell populations and also methods for maintaining a self-renewing state in culture. Conversely, in a therapeutic setting, even a small number of undifferentiated cells could result in tumor formation; therefore, we also need to understand how to prevent self-renewal of stem cells.
This proposed research will benefit the State of California and it citizens by addressing the molecular signals that are involved in triggering human embryonic stems cells to divide, die, or differentiate into neurons. Human embryonic stem cells have great potential to be used in cell replacement therapies once their growth and differentiation programs are understood. Our work studying the role of a major class of signaling molecules, Ephs and ephrins, is likely to shed light on how these signals are generated and responded to in culture. Understanding how this process is regulated at the molecular level may allow for the development of standard culture conditions that will optimize both self-renewal and homogeneity of cells; which in turn can lead to the large-scale production of stem cell populations and methods for maintaining a self-renewing state by reversible blocking of hESCs. It is also important to make sure that upon differentiation, no stem cells are left behind, because they may be able to give rise to tumors when put into the body.
The 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.
In 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.