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Abstract
Dissociated cells from embryonic day 14 mouse hypothalamus were infected with a retroviral construct containing the temperature sensitive variant 58 (tsA58) of the large T antigen (T-ag) of the Simian Virus 40 and the neomycin gene $(neo)\sp{37}.$ Two weeks after infection a $neo\sp+$ colony was selected, named tsV1 cells. The expression of the T-ag in these cells was confirmed by Southern blotting and immunocytochemistry techniques. This cell line contained cells with two different morphologies: the vast majority of cells were flat and stellate, and a smaller number were phase-bright and round cells. Southern Blotting and single cloning experiments suggested that the tsV1 cells were clonal in origin. Immunocytochemical and flow cytometric analyses indicated that the flat cells expressed typical glial markers including GFAP, NCAM, laminin fibronectin, S100, thrombospondin (TBS), and tenascin. After switching to the restrictive temperature (39$\sp\circ$C), there was a decrease in TBS and tenascin levels. The tsV1 cells support neuronal adhesion and neurite outgrowth of embryonic hypothalamic neurons. Conditioned media from tsV1 cells at 33$\sp\circ$C and 39$\sp\circ$C as well as conditioned media from astrocytes have similar mitogenic effect upon actively dividing type-1 astrocytes. These data suggest that the tsV1 cells are likely to be astrocytes.
When the tsV1 cells enter mitosis the flat cells become phase-bright, round, and, in some cases, develop process. These cells have condensed chromosomes and also express many typical neuronal proteins, such as neurofilament (NF) 200, NF-160, NF-68, tau, microtubule-associated protein 2, neuron-specific enolase, and GAP-43. This suggests that the tsV1 cell line may be a glial cell line expressing neuronal specific proteins in a cell cycle dependent fashion. Alternately, these proteins may have a functional role in mitosis. We believe that this system is a good model to understand the control of cell fate, mitosis, and maturation of hypothalamic glial and neuronal cells.
