Strategies for Restoring Olfactory Neurogenesis

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(Revised Abstract) DESCRIPTION (provided by applicant): The maintenance of olfactory function in humans as in other animals depends on the persistence of neurogenesis in the olfactory epithelium throughout life. If the progenitor population is destroyed during the course of an injury to the olfactory epithelium, the tissue undergoes metaplasia and reconstitutes as respiratory epithelium instead. We have shown that the globose basal cells (GBCs), among which are both broadly potent and neuronal-committed progenitors, can be selectively isolated by FACS and will engraft in the epithelium of a new host after transplantation. Accordingly, transplantation may be a viable strategy for restoring the epithelium's capacity for neurogenesis. Progress in implementing that strategy depends on the availability of a sufficiently large population of progenitors. To that end we are proposing experiments to test whether the pool of progenitors can be expanded in vitro and still engraft and differentiate appropriately in vivo. Two Specific Aims will be pursued. First, GBCs will be selectively isolated from normal mice that ubiquitously express GFP and others that have been lesioned with methyl bromide gas shortly before harvest. In the later case, the effect of the lesion is activate olfactory stem cells and to bias the population toward multipotency. The GBCs will be cultured in defined media with a melange of growth factors that have been shown to drive proliferation and/or neurogenesis in heterogeneous explant or dissociated cultures. Second, GBCs will be conditionally immortalized by transfection with a retroviral vector that encodes a temperature sensitive version of the large T antigen oncogene from SV40. The differentiation of the resulting clonal cell lines will be assayed after switching to the non-permissive temperature. Both the expanded primary isolates and cell lines that differentiate well after inactivation of the oncogene will be transplanted in order to define their capacity for differentiation in vivo (including a determination of whether donor-derived neurons express odorant receptors). Successful completion of the Aims will; 1) clarify the nature of growth factor control on olfactory neurogenesis; 2) determine whether cellular replacement is a viable strategy for restoring neurogenesis; 3) generate a set of protocols and cellular reagents that are useful for further studies of progenitor cell capacity and neuronal differentiation.