Neuronal and Glial Markers in Immortalized Olfactory Cell Lines

Abstract

Olfactory neurons are replaced in the adult olfactory epithelium by division and differentiation of cells close to the basement membrane of the olfactory epithelium. Among the basal cells is a stem cell which undergoes an asymmetric division, giving rise to a new stem cell and a precursor cell. The precursor cell divides again symmetrically and there may be additional symmetrical divisions before the progeny leave the cell cycle to differentiate [1]. We report here the characterization of cell lines created by retroviral insertion of the n-myc proto-oncogene into dividing cells of the adult mouse olfactory epithelium [2]. We have approximately 60 cell lines generated by this procedure, many of them cloned by single cell micromanipulation. These clones are heterogeneous in morphology but they fall into three broad categories: class 1 are mainly flat, epithelial-like cells; class 2 are a mixture of epithelial-like cells, round cells, and bipolar cells with short processes; and class 3 are a mixture of very large epithelial-like cells and bipolar cells with very long processes. Due to the slow growth of the class 3 cell lines and the difficulty of cloning them, we have concentrated our analysis on the class 1 and class 2 cells. Expression of neural and non-neural proteins was studied with a combination of immunocytochemistry, Western analysis, and reverse transcriptase polymerase chain reaction (RT-PCR). All cell lines express vimentin and keratin. Class 1 cell lines failed to express any neural cell markers when grown under our standard growth conditions (10% fetal calf serum, Dulbecco’s modified Eagle’s medium (DMEM), 5% CO2). Class 2 cell lines expressed a variety of neuronal proteins (neurofilament, 13tubulin, MAP-2, NCAM), although not all cell lines expressed all markers. Some clonal cell lines expressed the intermediate filament protein of glial cells (GFAP) in addition to neurofilament.