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The primitive neuroectodermal tumors (PNET) comprise a class of malignant nervous system neoplasms that afflict children. These tumors consist of cells that are morphologically identical to the primitive neuroepithelial cells normally seen in early stages of neural embryogenesis, supporting the notion that PNET result from a disturbance in the process of normal neuronal or glial differentiation. In the central nervous system, PNET occur most commonly in the cerebellum (medulloblastomas), but only occasionally in the cerebral hemispheres. We report here the establishment and characterization of a new human cell line (PFSK) derived from a PNET from the cerebral hemisphere of a child. The growth characteristics of PFSK cells were typical of an immortalized, transformed cell line. Cytogenetic and molecular genetic studies showed that three different sublines were present. In one of these sublines, sequences from chromosome 17 had been lost during establishment in culture. Immunocytochemical studies showed that PFSK cells expressed nestin, an intermediate filament protein normally expressed by neuroepithelial stem cells during neurulation. The PFSK cells did not express antigens typically found in terminally differentiated neurons or glia, indicating that this tumor cell line might represent neuroepithelial stem cells prior to commitment to a neuronal or glial lineage.