Abstract
Monoclonal antibodies were used in indirect immunofluorescence and immunoblot studies to examine the expression of four different classes of intermediate filaments, namely, neurofilaments, glial filaments, cytokeratin, and vimentin, in NTERA-2 cl.D1 (NT2/D1) pluripotent human embryonal carcinoma (EC) cells, and in the neurons derived from these cells by differentiation induced with retinoic acid. In the EC cell cultures, grown in the absence of retinoic acid, cytokeratin was the predominant intermediate filament detected by immunofluorescence; only a few cells expressed vimentin, and none expressed glial filament protein or any of the three neurofilament proteins (NF195, NF170, and NF70). Immunoblot analyses of cytoskeletal extracts of these cells supported these data. Two days after exposure to retinoic acid, all three neurofilament subunits were detected in a few cells with a non- neuronal morphology and, by double indirect immunofluorescence, were observed to colocalize with cytokeratin. The number of neurofilament- positive cells increased with time after initial exposure to retinoic acid, and although 95% of these cells contained cytokeratin initially, less than 5% of the neurofilament-positive cells retained cytokeratin 2 weeks later. By this time, many of the cells expressing all three neurofilaments but no cytokeratin exhibited a neuronal morphology. Vimentin was evident in a large number of cells in the cultures, but it was not detected in the neurofilament-positive cells. Also, many of the neurofilament-negative cells continued to express cytokeratin. No cells expressing glial filament proteins were found. Immunoblot analysis of the differentiated cultures also revealed all three neurofilament subunits, and vimentin and cytokeratin, but no glial filament protein.(ABSTRACT TRUNCATED AT 250 WORDS)