The rapid progress in our ability to isolate different stem cell and progenitor cell populations from the central nervous system has opened up the door to promising new avenues for the diagnosis and therapy of diseases of the CNS. These advances depend critically on the use of specific markers that allow us to distinguish between both developmentally and functionally related cell types. Using the limited number of cell type specific markers currently available, it has been possible to identify a number of key principles governing lineage relationships within the CNS. However, lineage studies also suggest that a much greater degree of cell diversity exists, that lies beyond the purview of markers available today. Here we propose to systematically develop a library of markers that can be used to identify and purify distinct neural cell populations with unprecedented specificity. To achieve this, we will combine our ability to isolate CNS precursor populations with the power of Phage Display technology. Through in vitro selection of single chain antibody clones that bind to the surface of isolated neural cells, we will specifically select for reagents that can be used for expression analysis in live cells. This protocol will allow rapid screening of large numbers of clones while avoiding problems inherent to procedures requiring the immunization of animals. Our plan of research will focus on the isolation of markers labeling 2 specific CNS populations: early glial progenitors and distinct populations of astrocytes. There are currently no markers that allow a positive identification of distinct stages of glial precursor differentiation. Isolating such markers will allow us to discern between oligodendroglial and astroglial precursors, as well as between progressive states of lineage restriction and proliferative potential, as observed in glial precursors from different regions of the CNS. Similarly, while GFAP continues to be used as the 1 defining marker of astrocytes, it is clear that astrocytic phenotypes can vary dramatically between astrocytes found during development, in acute lesions or in glial scar tissue. Preliminary data using glial progenitors strongly support the feasibility of this novel approach. Lay statement: The proposed research aims to generate new tools that will allow a better identification of different types of stem cells and their offspring in the brain. These tools will also help in developing better diagnostics and treatments for diseases affecting the brain. ? ? ?
