DIFFERENTIATION IS INDUCED IN 3-DIMENSIONAL CULTURES OF BRAIN-CELLS IMMORTALIZED BY THE LAP MAMMALIAN REGULATORY SYSTEM

Immortalized neuroectodermal precursor cell lines were generated from mouse brain by the SV40 large T antigen expressed under the control of the LAP (lac activating protein) mammalian regulatory system. The LAP system permits the reversible expression of T antigen as a function o f the exogenous inducer, isopropyl-beta-D-thiogalactopyranoside. Immor talized cells can be stably maintained in an undifferentiated state in monolayer cultures. Cell lines expressed the early neurofilament-like protein nestin, but not markers characteristic for mature cells such as the neurofilament light protein and glial fibrillary acidic protein . Downregulating the LAP-controlled T antigen with isopropyl-beta-D-th iogalactopyranoside was not sufficient to induce differentiation. Howe ver, when cells formed three-dimensional aggregates, differentiation t o a neuronal phenotype occurred, indicating that cell-cell interaction plays an important role in their differentiation. Cells in aggregates did not proliferate, even in the presence of T antigen, suggesting th at an aggregation-induced signal to cease growth was dominant over the growth signal of T antigen. Further morphological differentiation was induced by basic fibroblast growth factor. These immortalized cells s hould facilitate molecular and cellular studies concerned with the mec hanism of commitment, fate determination, and mitotic arrest of neuron al precursor cells in the developing mammalian CNS. (C) 1995 Wiley-Lis s, Inc.